The authors have declared that no competing interests exist.
Conceived and designed the experiments: AC GS LK. Performed the experiments: MM STL BL LK BW. Analyzed the data: MM. Contributed to the writing of the manuscript: MM GS LK AC.
Despite many advances in AIDS research, a cure for HIV infection remains elusive. Here, we performed autologous hematopoietic stem cell transplantation (HSCT) in three Simian/Human Immunodeficiency Virus (SHIV)-infected, antiretroviral therapy (ART)-treated rhesus macaques (RMs) using HSCs collected prior to infection and compared them to three SHIV-infected, ART-treated, untransplanted control animals to assess the effect of conditioning and autologous HSCT on viral persistence. As expected, ART drastically reduced virus replication, below 100 SHIV-RNA copies per ml of plasma in all animals. After several weeks on ART, experimental RMs received myeloablative total body irradiation (1080 cGy), which resulted in the depletion of 94–99% of circulating CD4+ T-cells, and low to undetectable SHIV-DNA levels in peripheral blood mononuclear cells. Following HSC infusion and successful engraftment, ART was interrupted (40–75 days post-transplant). Despite the observed dramatic reduction of the peripheral blood viral reservoir, rapid rebound of plasma viremia was observed in two out of three transplanted RMs. In the third transplanted animal, plasma SHIV-RNA and SHIV DNA in bulk PBMCs remained undetectable at week two post-ART interruption. No further time-points could be assessed as this animal was euthanized for clinical reasons; however, SHIV-DNA could be detected in this animal at necropsy in sorted circulating CD4+ T-cells, spleen and lymph nodes but not in the gastro-intestinal tract or tonsils. Furthermore, SIV DNA levels post-ART interruption were equivalent in several tissues in transplanted and control animals. While persistence of virus reservoir was observed despite myeloablation and HSCT in the setting of short term ART, this experiment demonstrates that autologous HSCT can be successfully performed in SIV-infected ART-treated RMs offering a new experimental
While antiretroviral therapy (ART) can reduce HIV replication, it does not eradicate the virus from an infected individual. Replication-competent viruses persist on ART and our incomplete understanding of these viral reservoirs greatly complicates the generation of a cure for HIV. In this study we performed, for the first time, hematopoietic stem cell transplant (HSCT) in the established model of SIV infection of rhesus macaques (RM). The HSC originating from the bone marrow were collected before SIV infection. After SIV infection, RM were treated with ART for several weeks to reduce viral replication before performing a total body irradiation and a transplant with their own, pre-infection, stem cells. The irradiation eliminated 94–99% of the circulating CD4+ T-cells, the main cell target of HIV/SIV infection. A successful engraftment of the HSC was observed and blood viral reservoirs were drastically reduced. However, when ART was interrupted, a rapid rebound of plasma viremia was observed in two out of three transplanted RM indicating that the massive reset of the hematopoietic compartment was not sufficient to eliminate the total-body virus reservoir in the setting of short term ART. This model of HSCT in SIV-infected RM provides a new platform to investigate HIV eradication strategies.
The introduction of antiretroviral therapy (ART) has dramatically reduced the morbidity and mortality associated with HIV infection and AIDS. However, currently available ART requires life long treatment with significant potential side effects and a cost that places an inordinate burden on public health systems. While reduction of HIV viral loads below detectable limits is often achieved in ART-treated individuals, a treatment that can eradicate or functionally cure HIV infection remains elusive. Many studies indicate that the key obstacle to cure HIV infection is the presence of a persistent reservoir of latently infected cells that are not eliminated by ART
In 2009 it was reported that an HIV-infected individual with acute myelogenous leukemia treated with myeloablative chemotherapy and allogeneic hematopoietic stem cell transplant (HSCT) from a Δ
SIV infection of non-human primates, such as rhesus macaques (RMs) has been used for over two decades as an
Six RMs were included in this study. All six RMs were males with an average age of 4.2 years (
(A) Three RMs received G-CSF 50 mg/kg subcutaneously daily for six consecutive days prior to HSC collection by leukopheresis and cryopreservation of the collected cells. Two apheresis procedures were performed on each transplant recipient. After collection of pre-infection HSCs, RMs were infected i.v. with 10,000 TCID50 RT-SHIVTC. Starting at day 28 post-infection, RMs received ART daily. After 37 to 54 days on ART, the three experimental RMs underwent TBI (total dose of 1080 cGy), fractionated in three doses given on three consecutive days pre-transplant. On the two following days, the leukopheresis products were infused. ART was interrupted 40 to 75 days post-transplant. (B) Three control RMs were infected i.v. with 10,000 TCID50 RT-SHIVTC and received ART for the same period of time as in the transplanted animals. The three control RMs did not undergo TBI/autologous HSCT.
T1 | T2 | T3 | C1 | C2 | C3 | ||||
4.3 | 4.2 | 4.2 | 4.2 | 4.1 | 4.1 | ||||
M |
M | M | M | M | M | ||||
+ | + | + | − | − | − | ||||
Infusion 1 | 1.6×108 | 2.3×108 | 3.0×108 | n/a | n/a | n/a | |||
Infusion 2 | 3.0×108 | 6.6×108 | 5.3×108 | n/a | n/a | n/a | |||
n/a | n/a | n/a | |||||||
Infusion 1 | 5.3×105 | 7.3×105 | 1.2×106 | n/a | n/a | n/a | |||
Infusion 2 | 1.0×106 | 4.4×106 | 8.0×105 | n/a | n/a | n/a | |||
n/a | n/a | n/a | |||||||
Pre-transplant | 37 | 38 | 54 | n/a | n/a | n/a | |||
Post-transplant | 49 | 40 | 75 |
n/a | n/a | n/a | |||
M: male.
n/a: non applicable.
TNC: total nucleated cells.
PMPA was interrupted at day 36 post-transplant.
As shown in
(A) The levels of SHIV-RNA, expressed as copies/ml of plasma are shown for each individual animal. Dotted line represents the limit of detection of the assay. (B) Longitudinal assessment of the absolute numbers of circulating CD4+ T-cell expressed as cells per µl. Transplanted animals are depicted in red and controls in blue. Shaded area represents the period of ART treatment.
The myeloablative TBI resulted in a drastic reduction of the absolute count of blood cells including neutrophils, monocytes, lymphocytes and CD4+ T-cells (
(A) Comparison of the pre- and post-transplant absolute numbers of circulating neutrophils, monocytes, lymphocytes and CD4+ T-cell expressed as cells per µl. Pre-transplant time point is the final assessment prior to TBI (SHIV-infected, on ART). Post-transplant time point represents the nadir cell count observed during the eleven days following TBI. Longitudinal assessment of the absolute numbers of circulating (B) neutrophils and (C) platelets expressed as cells per µl. Transplanted animals are depicted in red, controls in blue. Shaded area represents the period of ART treatment. Yellow area represents the period of platelet transfusion support. The dotted lines indicate the minimum level of neutrophils or platelets used to define engraftment.
Following transplantation and engraftment, we observed a rapid increase in the absolute leukocyte count and a slower reconstitution of the circulating CD4+ T-cells (
Longitudinal assessment of the absolute numbers of white blood cells (A) and circulating CD4+ T-cells (B) expressed as cells per µl are shown for each individual animal. Transplanted animals are depicted in red, controls in blue. Shaded area represents the period of ART treatment.
A few blips of transient low-level viremia were observed in the plasma of the three transplanted animals immediately after TBI and HSC infusion and while still on ART (
(A) Longitudinal assessment of the level of SHIV-RNA, expressed as copies/ml of plasma. (B) Longitudinal assessment of cell associated SHIV-DNA. Plain lines represent the level of SHIV-DNA in PBMCs determined by PCR. The dashed lines represent the estimated level of SHIV-DNA in CD4+ T-cells calculated based on PBMC frequency of infection determined by PCR and the frequency of CD4 + T-cells in PBMC determined by flow cytometry. Shaded area represents the period of ART treatment. (C) SHIV-DNA levels determined by PCR at necropsy, in sorted peripheral CD4+ T-cells and expressed as copies/million cells. Lines are drawn at the geometric mean. Mann Whitney U test was used to determine significance. Transplanted animals are depicted in red, controls in blue. Grey dotted lines represent the limit of detection of the assay.
ART was interrupted after stem cell engraftment (between 78 and 128 days post-initiation,
Several tissues were collected at necropsy including ileum, jejunum, colon, rectum, superficial and mesenteric lymph nodes as well as tonsils. SHIV-DNA levels in cell suspensions obtained from these tissues were quantified by PCR. As shown in
SHIV-DNA levels expressed as copies/million cells obtained at necropsy from the ileum, jejunum, colon, rectum, superficial and mesenteric lymph nodes, and tonsils are shown for each individual animal. Transplanted animals are depicted in red, controls in blue. Estimated number of CD4+ T-cells per million cells in tissues at necropsy is indicated in
The apparent cure of HIV infection in the “Berlin patient”
The key findings of this study are the following: (i) autologous HSCT using apheresis products collected prior to infection is feasible in SHIV-infected RMs; (ii) as expected, the myeloablative TBI used for conditioning induced a massive reset of the lympho-hematopoietic compartment, consequently resulting in the depletion of 94.2–99.2% of circulating CD4+ T-cells; (iii) animals receiving autologous HSCT under ART exhibited a prompt and pronounced decline in the peripheral blood viral reservoir (with undetectable SHIV-DNA in PBMCs in two out of three RMs) and maintained undetectable SHIV-RNA viremia with the exception of a few minor blips; (iv) two of the three transplanted RMs showed a very rapid rebound of viremia after ART interruption; and (v) the third transplanted RM, who was sacrificed for clinical reasons at day fourteen post ART interruption, had no detectable virus in plasma, PBMCs, tonsils, and GI tract, low but detectable levels of SHIV-DNA in sorted peripheral CD4+ T-cells and lymph nodes, and moderate levels of SHIV-DNA in the spleen.
Due to many logistical challenges of this experiment we chose to conduct the study in a temporally compressed fashion, with 37–53 days of ART before autologous HSCT, and interruption of ART after hematopoietic reconstitution, rather than prolonged continuation of therapy. This study was therefore designed to determine the impact of myeloablative irradiation on the viral reservoir, rather than the impact of prolonged viral suppression in conjunction with myeloablation. It is therefore possible that a similarly designed study, in which ART is maintained for a significantly longer period both before and after autologous HSCT, would have a different outcome, possibly demonstrating a more dramatic effect of autologous HSCT on the persistent reservoir of latently infected cells. Moreover, we cannot rule our the possibility that the level of virus suppression achieved by the short-term ART regimen in this experiment might not be as complete as what is observed in HIV-infected individuals on long-term ART. In this model of SHIV-infected RM, 5 to 7 weeks on ART pre-transplant may have been insufficient to fully suppress viral replication and the transient low-level viremia observed immediately post-transplant could be attributed to an insufficient period of ART pre-transplant. However, similar viral blips were observed in one patient who received allogeneic stem cell transplant after many years on combined ART
The myeloablative TBI used for conditioning resulted in the depletion of 94.2–99.2% of circulating CD4+ T-cells. Unfortunately, due to the clinical challenges of this innovative experiment, no tissue biopsies could be obtained immediately post-transplant to evaluate the TBI-induced CD4+ T-cell depletion in tissues. However, this study shows that myeloablative TBI and autologous HSCT did not prevent a rebound of viremia post-ART interruption in two out of three RMs despite relatively early ART initiation (day 28 post-infection). Moreover, while the SHIV-DNA level in PBMCs was undetectable or close to undetectable post autologous HSCT, it rapidly rebounded after ART interruption to levels that were similar or higher than those observed in the control animals at the same time-point. While in the third animal (T2) there was no sign of virus present in the plasma, PBMCs, and various tissues at the time of necropsy, this RM had to be sacrificed due to kidney failure at day fourteen after ART interruption making the interpretation of these data somewhat difficult. Of note, this study was not designed to identify the cellular and anatomic sources of the rapid plasma viral rebound observed in two transplanted RMs following ART interruption. Determining the relative contribution of tissue CD4+ T-cells, macrophages, and potentially other sources represents an important area for future investigation, amenable for interrogation with this model.
We acknowledge a number of limitations in our study including the small number of animals and the foreshortened time line involved. However, the demonstrated feasibility of this test-of-concept study in a non-human primate model of AIDS virus infection is per se an important result given the extreme complexity of the experimental protocol. The RMs included in this study underwent a series of procedures that have been only rarely, if ever, used in the same animal, including stem cell mobilization and harvesting by apheresis, RT-SHIV infection, daily four-drug ART administration, total body irradiation, re-infusion of HSCs, repeated platelet transfusions, and receipt of several antimicrobial prophylaxes. The feasibility of HSCT in SIV- or SHIV-infected RMs suggests, in our view, that further studies using this model in conjunction with longer term ART as well as additional interventions aimed at purging both the peripheral blood and lymphoid tissue-based viral reservoirs will provide critical information for the requirements to cure HIV infection in humans.
With respect to our understanding of the mechanisms responsible for “curing” HIV infection in the Berlin patient, our study supports the hypothesis that myeloablative TBI can cause a significant decrease in the viral reservoir in circulating PBMCs, even though it was not sufficient to eliminate all reservoirs. While the conditioning regimen in the Berlin patient also included antithymocyte globulin and chemotherapy, the use of a Δ
In conclusion, we have conducted the first test-of-concept study of myeloablative irradiation and autologous HSCT in ART-treated SHIV-infected RMs. This experiment demonstrated that autologous HSCT is a feasible intervention that can lead to a marked reduction of the virus reservoir in the peripheral blood, and can be used as an experimental
This study was conducted in strict accordance with USDA regulations and the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health, and were approved by the Emory University Institutional Animal Care and Use Committee (Protocol # YER-20000373-061714). SIV-infected animals were housed in standard non-human primate cages, received standard primate feed as well as fresh fruit and enrichment daily, and had continual access to water. Cages also contained additional sources of animal enrichment including objects such as perching and other manipulanda. Animal welfare was monitored daily. Appropriate procedures were performed to ensure that potential distress, pain, or discomfort was alleviated. The sedatives Ketamine (10 mg/kg) or Telazol (4 mg/kg) were used for blood draws and biopsies. Euthanasia of RMs, using Pentobarbital (100 mg/kg) under anesthesia, was performed only when deemed clinically necessary by veterinary medical staff and according to IACUC endpoint guidelines.
Six Indian RMs (
Autologous HSCs were harvested at two separate time points in each animal using our previously described apheresis procedure
The leukopheresis products were analyzed by flow cytometry prior to cryopreservation for the total nucleated cell dose, the CD34+ cell dose, CD3+ T-cell dose, CD4+ T-cell dose, CD8+ T-cell dose, and the CD20+ B-cell dose using the following antibodies; CD3 (clone SP34-2), CD34 (clone 563), CD45 (clone D058-1283), CD8 (clone RPA-T8) from BD Biosciences; CD20 (clone 2h7), CD4 (clone OKT4) from eBioscience.
The RMs were intravenously (i.v.) infected with 10,000 50% tissue culture infective doses (TCID50) of RT-SHIVTC. The virus stock was provided by Dr. Tom North (Emory University) and prepared as previously described
Efavirenz was provided by Bristol-Myers Squib, raltegravir was provided by Merck, and emtricitabine (FTC) and tenofovir (PMPA) were provided by Gilead Sciences. Efavirenz was fed at 200 mg per day by mixing the contents of a 200 mg capsule into food. Raltegravir was fed at 100 mg twice daily by mixing the drug into food. Stock solutions of FTC were prepared in phosphate-buffered saline (PBS, pH 7.4). PMPA was suspended in distilled water, with NaOH added to a final pH of 7.0. FTC and PMPA stocks were filter sterilized and stored at 4°C. These drugs were administered subcutaneously, at a dose of 30 mg/kg of body weight once daily. Drug dosages were adjusted weekly according to body weight.
The pre-transplant preparative regimen consisted of myeloablative TBI to a total dose of 10.8 Gy, given in three divided fractions of 3.6 Gy each (at a rate of 7.5 cGy/minute) using a Varian Clinac 23 EX (Varian). Irradiation took place on days −2, −1, and 0 (the day of transplant), with the final dose of irradiation given just prior to infusion of the first of two leukopheresis products.
Animals were treated with the following empiric antimicrobial agents in the peri-transplant period, as previously described
Transplanted animals received both platelet rich plasma and whole blood (irradiated at 2200 rad prior to transfusion) to treat thrombocytopenia (platelet count <50×106/ml) or anemia (hemoglobin <10 g/dl) or with the development of clinically significant bleeding. Blood product support adhered to ABO antigen matching principles.
EDTA-anticoagulated blood samples were collected regularly and used for a complete blood count, routine chemical analysis and immunostaining, with plasma separated by centrifugation within 1 h of phlebotomy. PBMCs were prepared by density gradient centrifugation. CD4+ T-cells were negatively selected from frozen PBMCs using magnetically labeled microbeads and subsequent column purification according to the manufacturer's protocol (Miltenyi Biotec). Tissue samples including ileum, jejunum, colon, tonsils and mesenteric and superficial lymph nodes were collected post-mortem. After two washes in RPMI and removal of connective and fat tissues, gut tissues were cut in small pieces and lymph nodes and tonsils were grinded using a 70-µm cell strainer. Gut cells were isolated by digestion with collagenase and DNase I for 2 h at 37°C and then passed through a 70-µm cell strainer. The cell suspensions obtained were washed and immediately used for immunostaining, cryopreserved or lysed in RLT+ buffer and stored at −80°C until use.
Plasma viral quantification was performed as described previously
Multicolor flow cytometric analysis was performed on whole blood or frozen PBMCs using predetermined optimal concentrations of the following fluorescently conjugated mAbs: CD3-PacBlue or -APC-Cy7 (clone SP34-2), CD95-PE-Cy5 (clone DX2), Ki-67-AF700 (clone B56), HLA-DR-PerCP-Cy5.5 (clone G46-6), CCR7-PE-Cy7 (clone 3D12), CCR5-PE or -APC (clone 3A9), CD45RA-FITC (clone L48), Biotin-CD122 (clone Mik-β3) from BD Biosciences; CD8-BV711 (clone RPA-T8), CD4-APC-Cy7 or -BV650 (clone OKT4), Streptavidin-PE from Biolegend, and CD28-ECD (clone CD28-2) from Beckman-Coulter. Flow cytometric acquisition and analysis of samples was performed on at least 100,000 events on an LSRII flow cytometer driven by the FACSDiva software package (BD Biosciences). Analyses of the acquired data were performed using FlowJo Version 10.0.4 software (TreeStar).
For the comparison of SHIV-DNA in sorted CD4+ T-cells in transplanted and control RMs the nonparametric Mann-Whitney U test was used. For the comparison of the proportion of memory CD4+ T-cells before and after transplant, a Wilcoxon matched-pairs signed rank test was used. Statistical significance was set at p<0.5. All analyses were performed using GraphPad Prism v4.0.
(PDF)
(PDF)
(PDF)
(DOCX)
We gratefully acknowledge Gilead, Bristol-Myers Squibb, and Merck for supplying the antiretroviral drugs, and T.W. North for providing the RT-SHIVTC. The authors also thank Stephanie Ehnert, Christopher Souder, and all the animal care and veterinary staff at the Yerkes National Primate Research Center.