HIV-1 Remission Following CCR5Δ32/Δ32 Haematopoietic Stem-Cell Transplantation Ravindra K

Letter https://doi.org/10.1038/s41586-019-1027-4

HIV-1 remission following CCR5Δ32/Δ32 haematopoietic stem-cell transplantation Ravindra K. Gupta1,2,3,4,5*, Sultan Abdul-Jawad1, Laura E. McCoy1, Hoi Ping Mok4, Dimitra Peppa3,6, Maria Salgado7, Javier Martinez-Picado7,8,9, Monique Nijhuis10, Annemarie M. J. Wensing10, Helen Lee11, Paul Grant12, Eleni Nastouli12, Jonathan Lambert13, Matthew Pace6, Fanny Salasc4, Christopher Monit1, Andrew J. Innes14,15, Luke Muir1, Laura Waters3, John Frater6,16, Andrew M. L. Lever4,17, Simon G. Edwards3, Ian H. Gabriel14,15,18,19 & Eduardo Olavarria14,15,19

A cure for HIV-1 remains unattainable as only one case has been a homozygous CCR5Δ32 (CCR5Δ32/Δ32) donor1. This 32-base-pair reported, a decade ago1,2. The individual—who is known as the deletion prevents CCR5 expression, rendering these cells resistant to ‘Berlin patient’—underwent two allogeneic haematopoietic stem-cell infection with HIV variants that interact with the CCR5 coreceptor8. transplantation (HSCT) procedures using a donor with a The only other case of a patient with HIV-1 who was transplanted homozygous mutation in the HIV coreceptor CCR5 (CCR5Δ32/ with CCR5Δ32/Δ32 cells and interrupted ART was the ‘Essen patient’9. Δ32) to treat his acute myeloid leukaemia. Total body irradiation In this case, ART was interrupted one week before allogeneic HSCT was given with each HSCT. Notably, it is unclear which treatment and rapid viral rebound of a pre-existing minority HIV-1 variant, which or patient parameters contributed to this case of long-term HIV was able to infect cells through the alternative CXCR4 coreceptor, was remission. Here we show that HIV-1 remission may be possible with observed three weeks later9,10. Such pre-existing CXCR4 variants were a less aggressive and toxic approach. An adult infected with HIV-1 not observed in the Berlin patient11. Three other cases transplanted underwent allogeneic HSCT for Hodgkin’s lymphoma using cells with wild-type CCR5 cells experienced viral rebound 12, 32 or 41 weeks from a CCR5Δ32/Δ32 donor. He experienced mild gut graft-versus- after ART interruption despite a considerable reduction of the HIV host disease. Antiretroviral therapy was interrupted 16 months reservoir12,13. after transplantation. HIV-1 remission has been maintained over We report an individual diagnosed with HIV-1 infection in 2003, a further 18 months. Plasma HIV-1 RNA has been undetectable with a CD4 nadir of 290 cells mm−3 and a baseline HIV-1 plasma viral at less than one copy per millilitre along with undetectable HIV-1 load of 180,000 copies per ml. ART was initiated with tenofovir diso- DNA in peripheral CD4 T lymphocytes. Quantitative viral outgrowth proxil fumarate, emtricitabine and efavirenz in 2012. assays from peripheral CD4 T lymphocytes show no reactivatable In December 2012, the individual was diagnosed with stage IVb virus using a total of 24 million resting CD4 T cells. CCR5-tropic, (nodular sclerosing) Hodgkin’s lymphoma. Hodgkin’s lymphoma was but not CXCR4-tropic, viruses were identified in HIV-1 DNA refractory to first-line chemotherapy (doxorubicin, bleomycin, vin- from CD4 T cells of the patient before the transplant. CD4 T cristine and dacarbazine) and a number of salvage regimens, including cells isolated from peripheral blood after transplantation did not etoposide, methylprednisolone, cytarabine and cisplatin; anti-CD30 express CCR5 and were susceptible only to CXCR4-tropic virus ex monoclonal antibody (Brentuximab) and mini-LEAM (lomustine, vivo. HIV-1 Gag-specific CD4 and CD8 T cell responses were lost etoposide, cytarabine and melphalan) were used. ART was switched after transplantation, whereas cytomegalovirus-specific responses to tenofovir disoproxil fumarate, emtricitabine and raltegravir during were detectable. Similarly, HIV-1-specific antibodies and avidities periods of chemotherapy for Hodgkin’s lymphoma; there was a 5-day fell to levels comparable to those in the Berlin patient following episode of ART interruption in late 2015 with an HIV-1 plasma viral transplantation. Although at 18 months after the interruption of load of 1,500 copies per ml that did not reach the viral set point. On the treatment it is premature to conclude that this patient has been cured, basis of resistance mutations K65R and M184V in reverse transcriptase these data suggest that a single allogeneic HSCT with homozygous as well as E157Q in integrase, the regimen was switched to rilpivir- CCR5Δ32 donor cells may be sufficient to achieve HIV-1 remission ine, lamivudine and dolutegravir; viral suppression was subsequently with reduced intensity conditioning and no irradiation, and the achieved. findings provide further support for the development of HIV-1 Mobilization of autologous peripheral blood stem cells failed despite remission strategies based on preventing CCR5 expression. the use of CXCR4 antagonists, thus precluding standard autologous The HIV-1 epidemic continues and nearly 37 million people are HSCT. Complete metabolic remission as confirmed by computed living with HIV-1 worldwide3. Although over 21 million people with tomography and positron-emission tomography criteria was achieved HIV-1 have access to lifelong antiretroviral therapy (ART)3, there is with ifosfamide, gemcitabine and vinorelbine (IGEV) chemotherapy considerable drug-resistant HIV in both untreated4 and treated5,6 indi- in March 2016. viduals in low- and middle-income countries and sustainability of ART An unrelated (nine out of ten) donor was identified from an interna- programmes is uncertain7. Drug-free durable HIV-1 suppression is tional registry with one allelic mismatch at HLA-B by high-resolution therefore an urgent global priority. HLA typing, who was CCR5Δ32/Δ32 (Extended Data Table 1 and Thus far, the only documented case of sustained HIV remission is Methods). No fully matched donors were identified in the registry. The the Berlin patient, who received two allogeneic HSCTs using cells from patient underwent conditioning with lomustine, cyclophosphamide,

1Division of Infection and Immunity, UCL, London, UK. 2Department of Infection, UCLH, London, UK. 3Mortimer Market Centre, Department of HIV, CNWL NHS Trust, London, UK. 4Department of Medicine, University of Cambridge, Cambridge, UK. 5Africa Health Research Institute, Durban, South Africa. 6Nuffield Department of Medicine, University of Oxford, Oxford, UK. 7IrsiCaixa AIDS Research Institute, Badalona, Spain. 8University of Vic – Central University of Catalonia (UVic-UCC), Vic, Spain. 9Catalan Institution for Research and Advanced Studies (ICREA), Barcelona, Spain. 10Translational Virology, Department of Medical Microbiology, University Medical Center, Utrecht, The Netherlands. 11Department of Haematology, University of Cambridge, Cambridge, UK. 12Department of Virology, UCLH, London, UK. 13Department of Haematology, UCLH, London, UK. 14Department of Clinical Haematology, Imperial College Healthcare NHS Trust, Hammersmith Hospital, London, UK. 15Imperial College London, London, UK. 16NIHR Oxford Biomedical Research Centre, Oxford, UK. 17Department of Medicine, National University of Singapore, Singapore, Singapore. 18Department of Haematology, Chelsea and Westminster Hospitals Foundation NHS Trust, London, UK. 19These authors contributed equally: Ian H. Gabriel, Eduardo Olavarria. *e-mail: [email protected]

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Full-donor chimerism was achieved in the whole-leukocyte and CD3+ 016) 2 T cell fractions from day 30 after transplantation and maintained in both cell fractions throughout (Fig. 1b). Host genotype was homozy- Δ (Feb 2019) gous wild-type CCR5 before allogeneic HSCT, and became CCR5 32/ 8 months off cART a Allo-HSCT100% (MaychimerismMild gut GvHD Stop CsA Stop cART (Sept 2017) 1 Δ32 after transplantation (Fig. 1c), with loss of CCR5 surface expres- TDF + FTC + RAL RPV + 3TC + DTG sion from circulating CD4 and CD8 T cells (Fig. 1d). At 180 days 10,000 600 after transplantation, cyclosporine-A was discontinued. Computed tomography and positron-emission tomography scans at 120 days and 1,000 400 365 days after transplantation confirmed complete metabolic remission with no subsequent relapse. Post-transplant white blood cell counts 100 200 and lymphocyte subsets returned to pre-transplant levels (Extended

<1.3 <0.9 <0.8 <0.8 CD4 count (cells per μl) Data Fig. 1), except for CD4 counts, which have been slower to recover

Viral load (copies per ml plasma ) 10 0 (Fig. 1a). –215 –141 –27 29 77 150 424 510 1,028 b 0 ART was maintained after HSCT and analytical treatment inter- 10,000 100 ruption was initiated at day 510 (September 2017). Weekly analyses cells) 6 1,000 of plasma viral load were performed for the first three months and monthly thereafter. HIV-1 plasma viral load remained undetectable 100 50 thereafter with a limit of detection of less than one copy of RNA per ml (Fig. 1a). Plasma concentrations of tenofovir disoproxil fumarate, 10 lamivudine and dolutegravir were negative by high-performance liq- Chimerism (% donor cells)

HIV DNA (copies per 10 1 0 uid chromatography at day 648 after transplantation, and a panel of –215 –141 –27 29 77 150 424 510 1,028 0 Time (days) all currently available antiretroviral drugs tested negative by liquid Haematological LACE chromatography mass spectrometry at 973 days after transplantation. treatment Anti- CD52 HIV-1 DNA associated with total peripheral blood mononuclear cells MTX CsA (PBMCs) fell below the limit of detection after transplantation (Fig. 1b). Total DNA in CD4 T cells at day 876 after transplantation was undetect- c d Pre-HSCT Post-HSCT day 72 Post-HSCT day 96 able in all replicates by ultra-sensitive qPCR (<0.65 HIV LTR copies

Δ 32 per million cells and <0.69 HIV-1 Gag copies per million cells) and 14 5 Δ 32 in 7 out of 8 replicates of the ultra-sensitive HIV-1 LTR ddPCR ; in t -SNE2 t -SNE2 t -SNE2

CCR5 WT CCR CCR5 WT/ NC Pre-HSCT Post-HSCT one replicate, a low-level signal was observed. Such occasional positive 15 t-SNE1 t-SNE1 t-SNE1 signals were also observed in the Berlin patient and may reflect a false Post-HSCT day 510 Post-HSCT day 819 ddPCR signal, potential contamination or evidence of very low levels CD8 of persistence of HIV-infected cells that either did not contain a fully CD4 NK total replication-competent virus or were unable to lead to recrudescence

t -SNE2 + t -SNE2 CD4 CCR5 given that the vast majority of target cells are incapable of being infected CD8 CCR5+ with the HIV CCR5-tropic variants of this patient (Fig. 2). HIV-1 DNA t-SNE1 t-SNE1 and RNA were also repeatedly undetectable in whole blood when tested Fig. 1 | Clinical course before and after allogeneic HSCT. with SAMBA II, a point-of-care isothermal amplification method (limit a, Antiretroviral treatment, and chemotherapy and immunosuppression of detection, 284 copies per ml; 95% confidence interval, 214–378 cop- associated with allogeneic HSCT along with plasma viral load (HIV-1 16 RNA) and CD4 count over time. Numbers below blue data points ies per ml) . Blood was obtained for a modified quantitative viral outgrowth indicate results of the ultrasensitive viral load assay. cART, combination 17,18 antiretroviral therapy. 3TC, lamivudine; CsA, cyclosporine-A; assay at 3 time points after HSCT on day 217 (on ART), days 678 DTG, dolutegravir; FTC, emtricitabine; LACE, lomustine, ara-C, and 876 (off ART) after transplantation. Analysis of the quantitative cyclophosphamide, etoposide; MTX, methotrexate; RAL, raltegravir; RPV, viral outgrowth assays indicated that HIV-1 virus was undetectable rilpivirine; TDF, tenofovir disoproxil fumarate. b, HIV-1 DNA in PBMCs on all occasions, giving an estimation of the reservoir in infectious and donor chimerism in T cell fraction. c, Genotyping of CCR5 alleles units per million resting CD4 T cells of less than 0.286, less than with agarose gel electrophoresis of PCR-amplified DNA fragments using 0.309 and less than 0.063, respectively. Pooling the results from the a 100-bp DNA ladder. NC, negative control. d, t-Distributed stochastic 3 quantitative viral outgrowth assays on a total of 24 million resting neighbour embedding (t-SNE) plots of PBMC before and after HSCT show CD4 T cells gives an estimate of less than 0.029 infectious units per that CD8 and CD4 CCR5 expression and cell populations change over time. NK, natural killer. a–d, These experiments were carried out once and million cells. sample size is n = 1 for all panels. We next sought to confirm that the post-transplant CD4 cells lacked expression of CCR5 and were resistant to HIV-1 infection (Fig. 2 and Extended Data Fig. 2). CD4 T cells from the study patient were chal- ara-C and etoposide followed by infusion of 3.6 × 106 CD34+ cells lenged in vitro with the CCR5-tropic viruses Ba-L and ZM247, and per kg. In vivo T cell depletion was achieved by anti-CD52 antibody productive HIV-1 infection over seven days was measured by (i) intra- (alemtuzumab), 10 mg daily for 5 days (days −7 to −3) before trans- cellular staining for HIV-1 p24 protein (Fig. 2a, b) and (ii) infectivity of plantation and graft-versus-host disease prophylaxis used cyclo- culture supernatants on indicator cell lines that respond to expression sporine-A with a short course of methotrexate. ART was continued of the HIV Tat protein (Extended Data Fig. 2). In contrast to an HIV- throughout with rilpivirine, lamivudine and dolutegravir (Fig. 1a). negative donor, post-transplant cells from the study patient could not Allogeneic HSCT was relatively uncomplicated and the patient was be infected with the CCR5-tropic viruses Ba-L and ZM247 (Fig. 2a, b). discharged on day 31 after transplantation. Reactivation of both The cells of the study patient were then challenged with the canonical Epstein–Barr virus and cytomegalovirus (CMV) occurred at day 85 CXCR4-tropic HIV-1 virus NL4-3. As expected, infection was observed after transplantation, requiring treatment with anti-CD20 monoclo- in cells and supernatants from both the patient and an HIV-negative nal antibody (rituximab) and ganciclovir, respectively. At day 77 after donor (control) (Fig. 2a, b and Extended Data Fig. 2). transplantation, the patient presented with fever and gastrointestinal To determine whether the study patient was infected with CCR5- or symptoms. Gastric, duodenal and colonic biopsies were consistent with CXCR4-using virus (or both), we deep-sequenced the V3 loop in HIV-1 grade 1 graft-versus-host disease, which resolved without intervention. envelope (the key determinant of coreceptor usage) and computational

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Standard ELISA abIndex patient Control 100 Ba-L b Control Off cART Gag+ Gag+ 80 0% 17.1% Index

1 60 Ba-L CCR5 10 a tropic Control

Infection (%) +– 40 Day 26 Day 423 Day 81 Day 965 1 20 Day 3Day 7 Allo-HSCT Signal:cut-off rati o LOD + Gag+ 100 ZM247 0 Gag gp160 0 200 400 600 800 1,000 0% 5.09% gp120 Time (days after allo-HSCT) ZM247 CCR5 tropic 10 c Detuned ELISA P68 40 P55 Off cART Infection (%) P51 30 1 gp41 Day 3Day 7 P40 Gag+ + Gag 20 LOD 12.3% 39.3% 100 NL4.3 P31 NL4.3 10 CXCR4 tropic 10 P24 Signal:cut-off rati o 0 Allo-HSCT 0 200 400 600 800 1,000 1

Infection (%) Time (days after allo-HSCT)

0.1 P18 1.0 Day 3Day 7 d Off cART 0.8 cdCCR5 U87 cells 250,000 0.6 LOD 200,000 Internal 150,000 0.4 * control RL U 100,000 Avidity index 50,000 0.2 0 110 100 1,000 0 Allo-HSCT Reciprocal dilution 0 200 400 600 800 1,000 Time (days after allo-HSCT) CXCR4 U87 cells * 250,000 Fig. 3 | HIV-specific antibodies. Humoral response dynamics were tested 200,000 150,000 at days 26, 423, 811 and 965 after HSCT (last two time points in absence

* RLU 100,000 of ART). a–d, Antibody levels were measured using western blot (a), the * 50,000 NL4-3 0 standard HIV-1 VITROS assay (b), a detuned low-sensitive version of the * 110 100 1,000 Ba-L Reciprocal dilution HIV-1 VITROS assay (c) and the limiting antigen avidity assay (d). Open D.A280 D.01CM_4412HAL symbols represent values under the limit of detection (LOD). Patient viruses 0.02 Dual tropic control virus a, c, d, Experiments were repeated twice independently with similar results. Fig. 2 | Susceptibility of the CD4 T cells of the index patient to CCR5- tropic and CXCR4-tropic HIV-1 viruses and coreceptor usage by viruses before HSCT. a, Representative plots of intracellular p24 Gag that expressed either CCR5 or CXCR4. As predicted from the genotype, staining within CD4 T cell populations three days after infection of robust infection was observed in CCR5- but not CXCR4-expressing isolated CD4 cells by CCR5-tropic viruses Ba-L and ZM247 and CXCR4- cells (Fig. 2d). tropic virus NL4.3. Numbers in the top left of the graph indicate the + Analyses of both antibody and T cell responses were carried out to percentage of infection (Gag ) of CD3 CD4 T cells, determined by p24 further investigate the absence of persistent HIV-1 infection and anti- staining. b, Percentage infection in CD3 CD4 T cells determined by p24 genic stimulation. Western blot analysis of antibodies demonstrated staining at day three and day seven after infection using CCR5- and CXCR4-tropic viruses. Data are mean ± s.e.m. and are representative of loss of p24, p31 and multiple other bands between pre- and post-transplant time points in pattern that is similar to patterns previously found three independent experiments each conducted in duplicate. Note, data 23 from the index patient are zero. c, Maximum likelihood phylogenetic in other transplanted patients who have remained on ART (Fig. 3a tree showing single genome env C2-V5 sequences (HXB2 env nucleotides and Extended Data Table 2), while antibodies against envelope protein 367–1,533) from PBMCs before HSCT. NL4.3, Ba-L and two subtype gp160 persisted and standard enzyme-linked immunosorbent assay D sequences from GenBank were also used. Red nodes indicate >70% (ELISA) for Env antibodies shows Env expression persisted (Fig. 3a, b). bootstrap support. gp120 amplicons from sequences marked with Low-sensitivity VITROS analysis and antibody avidity assays were an asterisk were cloned into a clade B gp160 Env plasmid. Virus env also consistent with loss of HIV antigens following allogeneic HSCT15 sequences marked by red asterisks generated infectious virus particles (Fig. 3c, d). CD8 and CD4 T cell virus-specific responses were deter- when co-transfected into HEK293T cells with the envelope-deficient Δ mined following stimulation with HIV-1 Gag- and CMV pp65-overlap- full-length HIV plasmid encoding luciferase (NL4-3 Env Luc). Black γ+ asterisks indicate sequences that did not generate infectious viruses. Scale ping peptide pools (Fig. 4a). Small Gag-specific IFN T cell responses bar, nucleotide substitutions per site. d, Pseudoviruses from c were used were identified by intracellular staining and CD4 multifunctional to infect U87 cells that express either CXCR4 or CCR5. RLU, relative responses (IFNγ, CD107a, TNF and IL-2) were detected before HSCT luminescence units. A dual tropic pseudovirus (WEAU-d15.410.787) (day −35) but not at days 72, 96, 510 and 819 after the procedure was produced in parallel as a positive control for infection (red line). (Fig. 4a–c). No HIV-1-specific T cell responses were detected to Nef, a–d, Experiments were performed independently three times with similar Pol and Env peptide pools at days 96 and 819 after transplantation results (sample size n = 1 healthy control donor and n = 1 index patient (Extended Data Fig. 3). By contrast, CMV-specific T cell responses were for a and b; sample size n = 9 clones tested in d). detected both before and after HSCT, albeit at reduced frequencies after HSCT compared to before HSCT (Fig. 4a–c). Both antibody and T cell responses observed here are highly sim- algorithms predicted CCR5 tropism19,20. For phenotypic verification, ilar to observations in the Berlin patient1,15. Important similarities we first performed single-genome sequencing of gp120 from pre-trans- between the two cases were CCR5-tropic HIV-1 infection and receipt plant PBMCs by limiting-dilution PCR21,22 (Fig. 2c). These single of a CCR5Δ32/Δ32 transplant. Graft-versus-host disease prophylaxis genome gp120 amplicons were cloned into gp160 expression vectors was very similar and used standard regimens (cyclosporine-A and and used to generate virions that were pseudotyped with patient- mycophenolate mofetil compared to cyclosporine-A and methotrexate) derived envelope protein21 (Fig. 2d), before infection of indicator cells that in randomized studies have shown similar outcomes24. In both

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a UnstimulatedHIV-1 Gag CMV pp65 Unstimulated HIV-1 GagCMV pp65 Pre- HSCT

CD8

CD4 0.033 0.563 2.026 0.021 0.046 8.614

Day 72

0.022 0.027 0.937 0.013 0.006 0.648

Day 96

0.044 0.028 0.983 0.003 0.002 0.300

Day 510 0.018 0.015 0.971 0.008 0.007 0.441

Day 819 0.068 0.062 1.871 0.012 0.008 0.141

bcIFNγ IFNγ Pre-HSCT Pre-HSCT 2.0 8 g Day 72 Day 72 Day 96 Day 96 6 Day 510 1.5

Day 510 Ga IV-1

g Ga IV-1 H H Day 819 Day 819 Pre-HSCT 4 Pre-HSCT 1.0 65 65 Day 72 Day 72 Day 96 2 Day 96 0.5 Day 510 Day 510 CMV pp CMV pp Day 819 Day 819 0 0 CD107a + + + + + + + + – – – – – – – CD107a + + + + + + + + – – – – – – – IFNγ + +++––––––+++ + – IFNγ ++++––––––+++ + – IL-2 + + – – + + – – + + – – + + – IL-2 + + – – + + – – + + – – + + – TNF + – + – + – + – + – + – + – + TNF + – + – + – + – + – + – + – +

Any function

Any function Fig. 4 | HIV-1 Gag-specific and CMV-specific T cell responses. (unstimulated) for each assay. b, c, Heat maps of levels of protein a, Representative fluorescence-activated cell sorting plots showing expression of CD107a, IFNγ, IL-2 and TNF in CD8+ T cells (b) and CD4 percentage of virus-specific CD8 and CD4 T cells identified by T cells (c) in response to stimulation with HIV-1 Gag and CMV pp65 intracellular staining for IFNγ, following stimulation with HIV-1 Gag or peptides before and after HSCT at the indicated days after Boolean gating. CMV pp65 peptide pools before and after HSCT at indicated days Functions are listed beneath the heat maps with each of their respective (72, 96, 510 and 819 after HSCT). A negative control containing combinations or any function detected. a, Experiment performed once PBMCs from the same subject but without peptide mix was included owing to limited cell numbers. cases, there was probable mild graft-versus-host disease, which may In terms of a way forward, we suggest that CCR5 gene-therapy strate- have contributed to the loss of HIV-1 infected cells. Finally, both gies using stem cells could conceivably be a scalable approach to remis- achieved and maintained full-donor chimerism in peripheral blood sion. As considerable graft-versus-host effects are probably important, that might have contributed to reduced reservoir size23,25. manifesting as early and sustained full donor chimerism in T cells, an Notable differences were that before allogeneic HSCT, our patient autologous approach needs to achieve high levels of replacement by was homozygous CCR5 wild-type compared to the heterozygous CCR5-depleted cells. Depletion of the CD4 CCR5 T cells and other CCR5Δ32/WT genotype observed in the Berlin patient. Our patient potential HIV reservoirs is a major challenge given the toxicities associ- received a reduced intensity conditioning regimen, which consisted ated with anti-thymocyte globulin and anti-CD52 antibody treatments exclusively of chemotherapy agents with known activity against lym- and the unknown role of anti-proliferative agents such as cyclosporin phoma26, whereas the Berlin patient received total body irradiation or methotrexate in clearing infected cells. in conjunction with cyclophosphamide as the conditioning regimen. This report demonstrates that the Berlin patient was not an anomaly, For lymphodepletion, our patient received the anti-CD52 antibody that remission of HIV infection can be achieved with reduced intensity campath whereas the Berlin patient was treated with anti-thymocyte drug regimens, that a single CCR5Δ32/Δ32 allogeneic HSCT is suffi- globulin. Our patient was treated with a short course of anti-CD20 cient and that total body irradiation is not required. Our observation antibody for reactivation of Epstein–Barr virus. There has been recent supports the development of HIV cure strategies based on preventing interest in the potential for anti-B cell therapy to disrupt B cell folli- the expression of the CCR5 coreceptor. cles that are known to contain persistent HIV in lymphoid tissues27, although we think this mechanism is unlikely to be related to the Online content remission observed here. Finally, this patient achieved full remission Any methods, additional references, Nature Research reporting summaries, source after a single allogeneic HSCT whereas the Berlin patient experienced data, statements of data availability and associated accession codes are available at a relapse of acute myeloid leukaemia and received further chemother- https://doi.org/10.1038/s41586-019-1027-4. apy with an anti-CD33 monoclonal antibody conjugate before a second Received: 7 February 2019; Accepted: 26 February 2019; allogeneic HSCT. Published online xx xx xxxx.

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1. Hütter, G. et al. Long-term control of HIV by CCR5 Delta32/Delta32 stem-cell 24. Hamilton, B. K. et al. Long-term follow-up of a prospective randomized trial transplantation. N. Engl. J. Med. 360, 692–698 (2009). comparing CYA and MTX with CYA and mycophenolate mofetil for GVHD 2. Allers, K. et al. Evidence for the cure of HIV infection by CCR5Δ32/Δ32 stem prophylaxis in myeloablative sibling donor hematopoietic cell transplantation. cell transplantation. Blood 117, 2791–2799 (2011). Bone Marrow Transplant. 48, 1578–1580 (2013). 3. UNAIDS. UNAIDS Data 2017 http://www.unaids.org/en/resources/ 25. Duarte, R. F. et al. CCR5 Δ32 homozygous cord blood allogeneic transplantation documents/2017/20170720_Data_book_2017 (2017). in a patient with HIV: a case report. Lancet HIV 2, e236–e242 (2015). 4. Gupta, R. K. et al. HIV-1 drug resistance before initiation or re-initiation of 26. Pavlů, J. et al. LACE-conditioned autologous stem cell transplantation for frst-line antiretroviral therapy in low-income and middle-income countries: a relapsed or refractory difuse large B-cell lymphoma: treatment outcome systematic review and meta-regression analysis. Lancet Infect. Dis. 18, 346–355 and risk factor analysis from a single centre. Hematol. Oncol. 29, 75–80 (2018). (2011). 5. The TenoRes Study Group. Global epidemiology of drug resistance after 27. Bronnimann, M. P., Skinner, P. J. & Connick, E. The B-cell follicle in HIV infection: failure of WHO recommended frst-line regimens for adult HIV-1 infection: barrier to a cure. Front. Immunol. 9, 20 (2018). a multicentre retrospective cohort study. Lancet Infect. Dis. 16, 565–575 (2016). 6. Barth, R. E., van der Loef, M. F., Schuurman, R., Hoepelman, A. I. & Wensing, A. M. Virological follow-up of adult patients in antiretroviral treatment Acknowledgements This study was funded by a Wellcome Trust Senior programmes in sub-Saharan Africa: a systematic review. Lancet Infect. Dis. 10, Fellowship in Clinical Science to R.K.G., research capability funding (RCF) 155–166 (2010). from UCLH BRC to R.K.G., as well as funding from Oxford and Cambridge 7. Avert. Funding for HIV and AIDS https://www.avert.org/professionals/ Biomedical Research Centres (BRC), amfAR (The Foundation for AIDS hiv-around-world/global-response/funding (2017). Research), through the amfAR Research Consortium on HIV Eradication 8. Simmons, G. et al. Primary, syncytium-inducing human immunodefciency (ARCHE) program (AmfAR 109858-64-RSRL), the MRC (MR/R008698/1 to virus type 1 isolates are dual-tropic and most can use either Lestr or CCR5 as L.E.M. and MRM008614/2 to D.P.). A.J.I. is supported by an NIHR Clinical coreceptors for virus entry. J. Virol. 70, 8355–8360 (1996). Lectureship, and acknowledges support from the NIHR and Imperial 9. Kordelas, L. et al. Shift of HIV tropism in stem-cell transplantation with CCR5 Biomedical Research Centre (BRC). E.N. received funding from the University Delta32 mutation. N. Engl. J. Med. 371, 880–882 (2014). College London Hospitals NHS Trust (UCLH)/University College London (UCL) 10. Verheyen, J. et al. Rapid rebound of a preexisting CXCR4-tropic human National Institute for Health Research (NIHR) Biomedical Research Centre and immunodefciency virus variant after allogeneic transplantation with CCR5 Δ32 research funding through an independent grant by ViiV Healthcare UK as part homozygous stem cells. Clin. Infect. Dis. 64, 684–687 (2019). of the EPIICAL Consortium. We thank the CHERUB (http://www.cherub.uk.net) 11. Symons, J. et al. Dependence on the CCR5 coreceptor for viral replication and IciStem Consortia (https://www.icistem.org/) for support and continuous explains the lack of rebound of CXCR4-predicted HIV variants in the Berlin discussion of results; N. Parmahand, M. Bandara, I. Jarvis, A. Fun, M. Lee, patient. Clin. Infect. Dis. 59, 596–600 (2014). L. Hedley, K. Ardeshna, A. Hill, N. Goel, R. Szydlo, D. Slade, S. Griffith and 12. Henrich, T. J. et al. Antiretroviral-free HIV-1 remission and viral rebound after C. Gálvez, Á. Hernández Rodríguez, V. González Soler and B. Rivaya Sánchez, allogeneic stem cell transplantation: report of 2 cases. Ann. Intern. Med. 161, E. van Maarseveen, L. Huyveneers, P. Schipper and D. de Jong; J. Apperley, 319–327 (2014). Z. Allwood and S. Loaiza and all the nurses in the BMT Unit that looked after 13. Cummins, N. W. et al. Extensive virologic and immunologic characterization in the patient. an HIV-infected individual following allogeneic stem cell transplant and analytic cessation of antiretroviral therapy: a case study. PLoS Med. 14, e1002461 Reviewer information Nature thanks Steven Deeks, Sarah J. Fidler, Timothy (2017). Henrich and the other anonymous reviewer(s) for their contribution to the peer 14. Bosman, K. J. et al. Development of sensitive ddPCR assays to reliably quantify review of this work. the proviral DNA reservoir in all common circulating HIV subtypes and recombinant forms. J. Int. AIDS Soc. 21, e25185 (2018). Author contributions R.K.G., S.A.-J., S.G.E., E.O., H.P.M. and I.H.G. conceived 15. Yukl, S. A. et al. Challenges in detecting HIV persistence during potentially and designed the study. S.A.-J., J.M.-P., A.M.J.W., M.S., M.N., F.S., L.M., I.H.G., curative interventions: a study of the Berlin patient. PLoS Pathog. 9, e1003347 L.E.M., D.P., H.P.M., P.G., E.N., A.M.L.L., L.W., S.G.E., M.P. and C.M. designed and/ (2013). or performed experiments. R.K.G., J.M.-P., A.M.J.W., S.A.-J., M.S., M.N., F.S., L.M., 16. Ritchie, A. V. et al. Performance evaluation of the point-of-care SAMBA I and II L.E.M., E.O., H.L., J.F., M.P., I.H.G., H.P.M., S.G.E., E.N., P.G., J.L., A.J.I. and C.M. HIV-1 Qual whole blood tests. J. Virol. Methods 237, 143–149 (2016). performed analyses and interpreted data. R.K.G., M.N., A.M.J.W., J.M.-P., A.M.L.L., 17. Laird, G. M. et al. Rapid quantifcation of the latent reservoir for HIV-1 using a J.L., H.P.M., and A.J.I. wrote the draft of the manuscript. J.M.-P., A.M.J.W., M.S., viral outgrowth assay. PLoS Pathog. 9, e1003398 (2013). M.N., F.S., S.A.-J., L.M., L.E.M., D.P., H.P.M., P.G., E.N., A.M.L.L., S.G.E., L.W., M.P., 18. Fun, A., Mok, H. P., Wills, M. R. & Lever, A. M. A highly reproducible quantitative I.H.G., H.P.M. and A.J.I. were involved in the critical revision of the manuscript for viral outgrowth assay for the measurement of the replication-competent latent important intellectual content. HIV-1 reservoir. Sci. Rep. 7, 43231 (2017). 19. Jensen, M. A., Coetzer, M., van ’t Wout, A. B., Morris, L. & Mullins, J. I. Competing interests The authors declare no competing interests. A reliable phenotype predictor for human immunodefciency virus type 1 subtype C based on envelope V3 sequences. J. Virol. 80, 4698–4704 Additional information (2006). Extended data is available for this paper at https://doi.org/10.1038/s41586- 20. Lengauer, T., Sander, O., Sierra, S., Thielen, A. & Kaiser, R. Bioinformatics 019-1027-4. prediction of HIV coreceptor usage. Nat. Biotechnol. 25, 1407–1410 (2007). Supplementary information is available for this paper at https://doi.org/ 21. Smith, N. M. et al. Proof-of-principle for immune control of global HIV-1 10.1038/s41586-019-1027-4. reactivation in vivo. Clin. Infect. Dis. 61, 120–128 (2015). Reprints and permissions information is available at http://www.nature.com/ 22. Watters, S. A. et al. Sequential CCR5-tropic HIV-1 reactivation from distinct reprints. cellular reservoirs following perturbation of elite control. PLoS ONE 11, Correspondence and requests for materials should be addressed to R.K.G. e0158854 (2016). Publisher’s note: Springer Nature remains neutral with regard to jurisdictional 23. Salgado, M. et al. Mechanisms that contribute to a profound reduction of the claims in published maps and institutional affiliations. HIV-1 reservoir after allogeneic stem cell transplant. Ann. Intern. Med. 169, 674–683 (2018). © The Author(s), under exclusive licence to Springer Nature Limited 2019

NAtUre| www.nature.com/nature RESEARCH Letter

Methods from the infection assay at a final dilution of 1/3 and 1/6. After a 48-h incubation, Data reporting. No statistical methods were used to predetermine sample size. cells were lysed by addition of Steady-Glo luciferase reagent (Promega) and lumi- The experiments were not randomized and the investigators were not blinded to nescence was read using a GloMax 96 Luminometer (Promega). For analysis, the allocation during experiments and outcome assessment. relative luminescence units were determined after the subtraction of background Patient consent pathway. Following the clinical decision in 2015 to under- luciferase activity from control wells containing cells (average of eight replicates) take allogeneic HSCT for treatment of aggressive lymphoma, the possibility of and subtraction of relative luminescence units from wells containing uninfected a CCR5Δ32/Δ32 match was discussed with the patient. International registry supernatant (average of six replicates). searches identified no fully matched (ten out of ten) donors. The only available Next-generation sequencing of HIV-1 Env V3 from genomic DNA. Nested PCR donors on the international registries were the selected nine out of ten match and was performed on isolated DNA to amplify the HIV-1 env region28. Subsequently, two eight out of tenmatches. The nine out of ten donor selected was therefore PCR products were cleaned using the QiaQuick PCR purification kit (Qiagen) the best available match, and subsequent testing confirmed that the potential and amplicons were sequenced using the MiSeq v.2 reagent kit (500 cycles) to donor was also homozygous for CCR5Δ32. Ultimately, because the best available yield paired-end reads of 250 bases each. Coreceptor tropism could be predicted donor happened to be CCR5 negative, there were no deviations from the standard by aligning reads to the consensus B sequence and isolating and trimming reads hospital consent protocol for HSCT, and therefore standard written informed that overlap the entire V3 region. Unique V3 sequences that are supported by consent for ‘LACE conditioned allogenic stem cell transplant’ was obtained from 1.75% were used for HIV-1 coreceptor tropism predicted using geno2pheno and the patient. PSSM algorithms. In December 2015, six months before HSCT, coreceptor usage was inferred Single-genome sequencing from PBMCs before HSCT. Single genomes were from sequencing of plasma RNA that reported CCR5-tropic virus. The potential generated as follows. Genomic DNA was extracted from total peripheral PBMCs for remission was then discussed with the patient and that a supervised treat- using the Qiagen DNeasy kit. Two rounds of nested PCR were then performed ment interruption would be needed to demonstrate successful remission. Ethical using previously validated primers and Invitrogen High Fidelity platinum Taq approval for treatment interruption, frequent HIV viral load monitoring and tissue polymerase. The 2-kb env amplicons were visualized by agarose gel electrophoresis sampling was sought using the standard NHS framework in mid to late 2016. and Sanger-sequenced using a primer set internal to env. The protocol specified that at 12 months after the transplant, if the viral load was Sequence alignment and phylogenetic tree construction. env amplicons consistently <50 copies per ml on treatment with ‘target not detected’ for at least generated by single-genome sequencing were Sanger-sequenced. Before alignment, 6 months on the 2 most recent consecutive visits, ART would be withdrawn and sequences were trimmed at the 5′ and 3′ ends until base calls consistently reached a thereafter viral load would be monitored weekly for the first 3 months, monthly for quality score ≥30, resulting in 99.9% accuracy of base calls. Patient env nucleotide a further 9 months if undetectable at all time points and every 3 months between sequences were aligned at the protein sequence level using MUSCLE v.3.8.3129 years 1 and 4. The protocol specified that if assays for the HIV reservoir were and mapped back to nucleotide, with minor manual adjustments. Sequences from deemed negative by a group of UK experts from CHERUB, we would thereafter laboratory isolates NL4.3 and Ba-L were included for reference, together with two monitor viral load every six months. The protocol stated that if viral rebound HIV-1 subtype D reference isolates from the Los Alamos HIV Sequence Database occurred, moderate or severe symptoms would not be expected owing to the (http://www.hiv.lanl.gov/) to serve as an outgroup. A maximum likelihood phy- likely low viral load in cases in which the donor chimerism is close to 100%. For logeny was estimated using RAxML v.830, with the general time-reversible nucle- the assessment and management of risk, the protocol stated that there was a risk otide substitution model and gamma-distributed rate heterogeneity. Stationary of rebound viraemia, symptoms such as fever and other flu-like symptoms and frequencies of nucleotides were estimated from their counts in the sequences. Clade that this would be mitigated by performing regular viral-load testing to detect support was estimated from 1,000 nonparametric bootstrap replicate datasets. The viral rebound early, restarting ART when we observe at least 2 viral loads above maximum likelihood phylogeny was rooted on the outgroup branch and visualized 200 copies per ml one week apart in the absence of any other cause of viral blips, using FigTree v.1.4.4 (http://tree.bio.ed.ac.uk/software/figtree/). and monitoring the percentage of CD4 cells at least monthly for the first year. UK Phenotypic determination of CCR5 or CXCR4 usage by patient-derived Env NHS Health Research Authority Research Ethics Committee approval was obtained clones. Selected gp120 amplicons were cloned into a gp160 expression vector in April 2017 with reference number 17/SW/0021, protocol number 16/0594. The and transfected into HEK293T cells with the envelope-deficient full-length HIV patient provided full written, informed consent in July 2017. The interruption did plasmid encoding luciferase (NL4-3 ΔEnv Luc). Pseudoviruses were collected, not occur until September 2017, 16 months after transplantation. The patient was filtered and titrated threefold in U87 cells expressing either CXCR4 or CCR5. registered to the IciStem consortium as IciStem#36. A dual tropic pseudovirus (WEAU-d15.410.787) was produced in parallel as a Testing of chimerism. We measured whole leukocyte and T-cell-specific (CD3 positive control for infection. After a 48-h incubation, cells were lysed by addition selected) chimerism by short tandem repeats analysis with the PowerPlex16 system. of Steady-Glo luciferase reagent (Promega) and luminescence was read using a Primary cell culture and CD4 isolation. PBMCs were isolated from whole blood GloMax 96 Luminometer (Promega). by centrifugation on a Lymphoprep (Axis-Shield) gradient. The cells collected at Modified quantitative viral outgrowth assay. The quantitative virus outgrowth the interface were washed with PBS and stimulated for 3 days in the presence of assay was performed as previously described18 with modifications. In brief, total PHA and IL-2 (10 pg ml−1) in RPMI 1640 supplemented with 10% FCS. Stimulated CD4 T cells were isolated from PBMCs by immune-magnetic negative selection PBMCs were used for negative selection of CD4 cells with antibody-coated mag- (StemCell Technologies). Cells displaying activation markers (CD25, CD69 and/or netic beads (Mojosort, Biolegend). HLA-DR) were labelled with FITC-conjugated antibodies (Biolegend) and depleted Extracellular receptor staining. Stimulated PBMCs were washed with FACS by FITC selection kit (StemCell Technologies). T cells with activation markers buffer (PBS and 1% FCS) then stained with viability dye (Ghost Dye Violet 450, adhering to the beads were recovered, additionally activated with phytohaemag- Tonbo Bioscience), followed by anti CD3–APC, anti CD4–FITC and anti CD195– glutinin-L (PHA-L, Sigma-Aldrich), seeded in a limiting dilution and co-cultured PE (also known as CCR5) (BioLegend). Cells were washed and fixed in 2% para- with SupT1-CCR5 for at least 21 days. formaldehyde. At least 105 lymphocytes were acquired on a BD LSRFortessa flow Resting CD4 T cells were recovered from the eluent and cultured in the presence cytometer and analysed using FlowJo (Tree Star). of 100 nM raltegravir and 20 nM efavirenz for 1–3 days to allow unintegrated viral Spreading infection. Infection of primary cells using full-length CCR5- and CXCR4- DNA to degrade. Afterwards, resting CD4 T cells were counted, activated with tropic HIV-1 viruses. CXCR4-tropic hybrid clone NL4-3 and CCR5-tropic viruses PHA-L, tenfold excess of irradiated allogeneic PBMCs and 10 units per ml IL-2, Ba-L and ZM247 were used to infect CD4 T-cells for 4 h (623, 1,150 and 430 pg and seeded in a limiting dilution. The activation mix was washed away 24 h later per well, respectively), before cells were washed twice and incubated in RPMI 1640 and SupT1-CCR5 cells were added to each culture well. culture medium supplemented with 10% FCS and IL-2 (10 pg ml−1) for a period The dilution series include replicates at 2.5 million, 0.5 million and 0.1 million of 7 days. Culture medium was changed on day 3 and the collected supernatants CD4 T cells per well as appropriate. Cultures were maintained for at least 21 days. were stored for analysis. Cells were stained with viability dye (Ghost Dye Violet 450, Cells were monitored for syncytia formation, and the supernatant was sampled Tonbo Bioscience) then fixed in 4% paraformaldehyde, permeabilized (BD Perm/ regularly for p24. The infectious units per million cells were calculated by limiting Wash, BD Biosciences) and stained for intracellular p24 using anti-p24 KC57-RD1 dilution statistics. conjugated antibody (Beckman Coulter). The percentage of infected cells was mon- Reagents for the quantitative viral outgrowth assay. IL-2 was obtained from the itored by flow cytometry using BD LSRFortessa flow cytometer (BD Biosciences) Centre for AIDS reagents, National Institute of Biological Standards and Control and analysed by FlowJo software (Tree Star). ZM2471 was a gift from B. Hahn. (NIBSC). Raltegravir was obtained through the NIH AIDS Reagent Program, Supernatant infectivity assay on HeLa TZM-bl reporter cells. In this assay, infectivity Division of AIDS, NIAID, NIH: raltegravir (11680) from Merck. Efavirenz was was measured as Tat-induced gene expression of the firefly luciferase reporter also obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, after a single round of virus infection. In brief, 104 TZM-bl cells per well were NIH: efavirenz. seeded in a 96-well white plate in DMEM culture medium supplemented with Residual viraemia by single copy assay (ultra-sensitive viral load assay). Residual 10% FCS for 24 h. After this, medium was replaced with supernatant collected viraemia (HIV RNA) was measured by ultracentrifugation of up to 6.5 ml of plasma Letter RESEARCH

(4–6.5 ml) at 170,000g at 4 °C for 30 min, followed by viral RNA extraction using Fortessa X20 using BD FACSDiva8.0 (BD Bioscience) and analysed using FlowJo the m2000sp Abbot RealTime HIV-1 Assay device and laboratory-defined appli- 10 (Tree Star). t-SNE analysis was performed using the mrc.cytobank platform. cations software from the instrument. HIV-1 RNA copies in the low range were Intracellular cytokine staining. PBMCs were thawed and resuspended in 3 determined by an in-house calibration curve set (range, 10–10 absolute copies), RPMI complete medium. After overnight rest at 37 °C and 5%CO2, PBMCs were which had previously been validated31 using a standard HIV-1 DNA control from stimulated for 6 h with 2 μg ml−1 HIV-1 Gag pools or CMV pp65 (JPT Peptide the WHO in the range of 128–0.5 copies per ml. Limit of detection was calculated Technologies) in the presence of 1 μg ml−1 anti-CD28 and anti-CD49d CoStim relative to the plasma volume used in each sample. antibodies (BD Biosciences), 2 μM Monensin (BD Biosciences), 10 μg ml−1 brefel- Quantification of HIV antibodies. Specific HIV-1 antibodies in longitudinal din A (Sigma-Aldrich) and anti-CD107a–BV605 (Biolegend). Where indicated and serum samples were tested in a qualitative western blot assay (New Lav Blot I, cell numbers permitted, PMBCs were stimulated with 2 μg ml−1 HIV-1 Env, Pol Bio-Rad). Standard and low-sensitive versions of the VITROS anti-HIV-1 assay and Nef peptide pools. Following stimulation, virus-specific T cells were identified (Ortho-Clinical Diagnostics) as well as the limiting avidity antigen assay were by intracellular cytokine staining as previously described. In brief, cells were fixed measured in same samples as previously described2. In brief, four recombinant and permeabilized (CytoFix/CytoPerm BD Biosciences) followed by intracellu- antigens (HIV-1 Env 13, HIV-1 Env 10, HIV-1 p24 and HIV-2 Env AL) derived lar cytokine staining for IFNγ–PE–Cy7, TNF–FITC and IL-2–PercP eFluor710. from HIV-1 core, HIV-1 envelope and HIV-2 envelope proteins were quantified. Stimulation with 0.005% DMSO in the presence of costimulatory antibodies, pro- The optimized version of the LS-VITROS assay (detuned or low-sensitive version) tein transport inhibitors and CD107a was performed as a negative control. uses a 1:400 dilution of an HIV-positive sample. The cut-off was set to a ratio of Reporting summary. Further information on research design is available in signal:cut-off of 20. The avidity assay measures the capacity of the guanidine to the Nature Research Reporting Summary linked to this paper. elute low-avidity and low-affinity antibodies after antigen–antibody bonds have formed. The results are reported as an avidity index, which was calculated as a Data availability ratio of the ratio of signal:cut-off of the sample incubated in guanidine to the ratio SGA sequences are available via GenBank under accession numbers MK493056– of signal:cut-off of the sample incubated in PBS. Cut-off was established at 0.51. MK493075. Source Data are available for Figs. 1–4. Flow cytometry. The following fluorochrome-conjugated antibodies were used in this study: CD14–BV510, CD19–BV510, CD3–APC Fire 750 or CD3–BV605, 28. Symons, J. et al. Impact of triplicate testing on HIV genotypic tropism prediction CD4–PE/Dazzle 594, or CD4–APC Fire 750, CD8–BV421, CCR5–PE/Dazzle in routine clinical practice. Clin. Microbiol. Infect. 18, 606-612 (2012). 594, CD56–PE–Cy7 (Biolegend) for surface antigens; and IFNγ–PE–Cy7 (BD 29. Edgar, R.C. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 32, 1792–1797 (2004). Biosciences), TNF–FITC (Biolegend) and IL-2–PercP eFluor710 (eBioscience) 30. Stamatakis, A. RAxML version 8: a tool for phylogenetic analysis and for intracellular staining. PBMCs were washed in PBS and surface-stained at 4 °C post-analysis of large phylogenies. Bioinformatics 30, 1312–1313 (2014). for 20 min with saturating concentrations of different combinations of antibodies 31. Amendola, A. Standardization and performance evaluation of “modifed” and in the presence of fixable live/dead stain (Invitrogen). Cells were then fixed and “ultrasensitive” versions of the Abbott RealTime HIV-1 assay, adapted to permeabilized for detection of intracellular antigens. Cells were acquired on a BD quantify minimal residual viremia. J. Clin. Virol. 42, 17–22 (2011). RESEARCH Letter

Extended Data Fig. 1 | Blood cell populations over time. 3TC, lamivudine; CsA, cyclosporine-A; DTG, dolutegravir; RPV, rilpivirine; WBC, white blood cells. Letter RESEARCH

Extended Data Fig. 2 | Susceptibility of CD4 T cells from the index infected with CCR5- and CXCR4-tropic viruses were collected on days patient to CCR5-tropic and CXCR4-tropic HIV-1. a, Experimental flow three and seven to measure infectivity on HeLa TZM-bl reporter cells. for measurement of infection by intracellular p24 Gag staining. Control Infectivity is measured as a reduction in the gene expression of the cells were from a healthy HIV−CCR5 donor. b, Flow cytometry analysis Tat-induced firefly luciferase reporter in TZM-bl cells. data are of PBMCs following three days of stimulation exhibiting the expression mean ±s.e.m. n = 2: one donor and one index patient. Experiments were pattern of CCR5 receptor within CD3 CD4 T cells in both healthy donor repeated three times with similar results. (control) and index patient. c. Culture supernatants from CD4 T cells RESEARCH Letter

Extended Data Fig. 3 | CD8 T cell responses and CD4 T cell responses to identified by intracellular staining for IFNγ, following stimulation with HIV. Representative fluorescence-activated cell sorting plots showing the HIV Pol, Env and Nef peptide pools after HSCT at days 96 and 819. percentage of virus-specific CD8 T cells (top) and CD4 T cells (bottom) Letter RESEARCH

Extended Data Table 1 | Comparison of blood group and tissue type between stem cell donor and index case RESEARCH Letter

Extended Data Table 2 | Detection of bands on western blot pre- and post-transplantation nature research | reporting summary

Corresponding author(s): Ravindra K Gupta

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Antibodies Antibodies used The following fluorochrome-conjugated antibodies were used in this study: CD14 BV510, CD19 BV510, CD3 APC Fire 750 or CD3 BV605, CD4 PE/Dazzle 594, or CD4 APC Fire 750, CD8 BV421, CCR5 PE/Dazzle 594, CD56 PeCy7 (Biolegend) for surface antigens; and IFN- PeCy7, TNF- FITC (Biolegend) and IL-2 PercP eFluor710 (eBioscience)

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Eukaryotic cell lines Policy information about cell lines Cell line source(s) TZMBL from NIH AIDS Reagent repository; HEK 293T cells. U87 CD4+CCR5+ Cells (Catalog Number 4035) and U87 CD4+CCR5+ Cells (Catalog Number 4036) were obtained from the NIH AIDS reagents program. 293T/17 [HEK 293T/17] cells for pseudovirus production were obtained from ATCC (CRL-11268).

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Commonly misidentified lines The ATCC stock of the parental cell line U87 MG has been shown to be different to the original U87 stock generated at the (See ICLAC register) University of Uppsala Sweden. The U87 cells used in this study were derived from the original Swedish line not the ATCC line as documented by the NIH AIDS reagents program. These are the gold standard cells for determining co-receptor use of HIV viruses.

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Recruitment This is extremely rare and is a case report that had full ethical approval for analytical treatment interruption

Ethics oversight Ethical approval for this study ‘HIV treatment interruption following Stem cell transplantation with CCR5 negative donor cells for haematological malignancy’ was obtained from the UK Health Research Authority Research Ethics Committee reference number 17/SW/0021, protocol number 16/0594. Written informed consent was obtained from the individual.

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Data collection from Dec 2015 to Feb 2019

Outcomes time to viral rebound, reservoir size by QVOA, PCR on CD4 T cells, CD4 trends, cancer remission

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Field strength Specify in Tesla

Sequence & imaging parameters Specify the pulse sequence type (gradient echo, spin echo, etc.), imaging type (EPI, spiral, etc.), field of view, matrix size, slice thickness, orientation and TE/TR/flip angle.

Area of acquisition State whether a whole brain scan was used OR define the area of acquisition, describing how the region was determined.

Diffusion MRI Used Not used

Preprocessing Preprocessing software Provide detail on software version and revision number and on specific parameters (model/functions, brain extraction, segmentation, smoothing kernel size, etc.).

Normalization If data were normalized/standardized, describe the approach(es): specify linear or non-linear and define image types used for transformation OR indicate that data were not normalized and explain rationale for lack of normalization.

Normalization template Describe the template used for normalization/transformation, specifying subject space or group standardized space (e.g. original Talairach, MNI305, ICBM152) OR indicate that the data were not normalized.

Noise and artifact removal Describe your procedure(s) for artifact and structured noise removal, specifying motion parameters, tissue signals and physiological signals (heart rate, respiration).

Volume censoring Define your software and/or method and criteria for volume censoring, and state the extent of such censoring.

Statistical modeling & inference Model type and settings Specify type (mass univariate, multivariate, RSA, predictive, etc.) and describe essential details of the model at the first and second levels (e.g. fixed, random or mixed effects; drift or auto-correlation).

Effect(s) tested Define precise effect in terms of the task or stimulus conditions instead of psychological concepts and indicate whether ANOVA or factorial designs were used.

Specify type of analysis: Whole brain ROI-based Both

Statistic type for inference Specify voxel-wise or cluster-wise and report all relevant parameters for cluster-wise methods. (See Eklund et al. 2016)

Correction Describe the type of correction and how it is obtained for multiple comparisons (e.g. FWE, FDR, permutation or Monte Carlo).

Models & analysis n/a Involved in the study Functional and/or effective connectivity Graph analysis Multivariate modeling or predictive analysis

Functional and/or effective connectivity Report the measures of dependence used and the model details (e.g. Pearson correlation, partial correlation, mutual information).

Graph analysis Report the dependent variable and connectivity measure, specifying weighted graph or binarized graph, subject- or group-level, and the global and/or node summaries used (e.g. clustering coefficient, efficiency, etc.).

Multivariate modeling and predictive analysis Specify independent variables, features extraction and dimension reduction, model, training and evaluation metrics. October 2018