Abstract
This study tested whether donor-derived HIV-specific immune responses could be detected when viral replication was completely suppressed by the continuous administration of highly active antiretroviral therapy (HAART). A regimen of fludarabine and 200 cGy total body irradiation was followed by infusion of allogeneic donor peripheral blood cells and posttransplantation cyclosporine and mycophenolate mofetil. Viral load, lymphocyte counts, and HIV-1–specific CD8+ cell immune responses were compared before and after hematopoietic cell transplantation (HCT). Uninterrupted administration of HAART was feasible during nonmyeloablative conditioning and after HCT. The HIV RNA remained undetectable and no HIV-associated infections were observed. CD8+ T-cell responses targeting multiple epitopes were detected before HCT. After HCT a different pattern of donor-derived HIV-specific CTL responses emerged by day +80, presumably primed in vivo. We conclude that allogeneic HCT offers the unique ability to characterize de novo HIV-1–specific immune responses. This clinical trial was registered at ClinicalTrials.gov (identifier: NCT00112593).
Introduction
Recent studies have shown that autologous hematopoietic cell transplantation (HCT) is feasible for treatment of HIV-associated lymphoma when HIV is controlled with highly active antiretroviral therapy (HAART).1 This study investigates a nonmyeloablative (NM) HCT regimen, NMHCT,2 combined with uninterrupted HAART to determine the effect on the control of HIV replication and on the development of HIV-1–specific immune responses.
Methods
Study population
Eligibility included HIV-1–infected patients with hematologic malignancies on suppressive HAART. The transplantation protocol was approved by the Institutional Review Board of the Fred Hutchinson Cancer Research Center (FHCRC). Written informed consent was obtained in accordance with the Declaration of Helsinki.
Transplantation
The preparative regimen, 90 mg/m2 total dose of fludarabine and 200 cGy total body irradiation, was administered as described.2 Patients received granulocyte colony-stimulating factor (G-CSF)–mobilized peripheral blood stem cells (PBSCs) from an HLA allele–matched related or unrelated donor. Prophylaxis for graft-versus-host disease (GVHD) consisted of cyclosporine (CSP) and mycophenolate mofetil (MMF).2,3 HAART was given through conditioning and after HCT (Table 1).
Table 1.
Patient characteristics and transplantation outcomes
| Patient 1 | Patient 2 | |
|---|---|---|
| Age/sex | 39/M | 33/M |
| Diagnosis | AML | AML |
| CMV R/D | +/− | −/− |
| Donor | Unrelated | Sibling |
| Patient and donor HLA | ||
| A | 2402, 0201 | 0101, 2601 |
| B | 2705, 3901 | 0801, 3502 |
| C | 0102, 1203 | 0701, 0401 |
| DRB 1 | 0701, 1601 | 0301, 1104 |
| DQB 1 | 0201, 0502 | 0201, 0301 |
| Cell dose/kg recipient | ||
| CD34+, ×106 | 4.58 | 10.63 |
| CD3+, ×108 | 1.66 | 2.83 |
| HAART regimen | Efavirenz, abacavir, tenofovir | Efavirenz, abacavir, lamivudine |
| GVHD prophylaxis | CSP/MMF | CSP/MMF |
| Relapse (treatment) | Cytogenetic relapse, day +44 (CSP/MMF withdrawn) | None |
| GVHD (treatment) | Grade II skin/gut, day +56 (Pred/MMF/FK/B-B) BO, day +254 (Pred/Immuran/MMF/FK) | None |
| Survival | Died, day +301 | > 180 days |
| Cause of death | GVHD + BO | NA |
| HIV-related illness | None | None |
| HIV RNA (copies/mL) | ||
| Pre | < 30 | < 30 |
| Day +14 | < 30 | < 30 |
| Day +28 | < 30 | < 30 |
| Day +56 | < 30 | < 30 |
| Day +100 | < 30 | < 30 |
| Day 180 | < 50 | < 30 |
| HIV proviral DNA | ||
| Pre | Positive | Positive |
| Day +28 | Positive | Positive |
| Day +56 | Negative | Positive |
| Day +100 | Negative | Positive |
| HIV culture | ||
| Pre | Not isolated | Not isolated |
| Day +14 | Not isolated | Not isolated |
| Day +28 | Not isolated | Not isolated |
| Day +56 | Not isolated | Not isolated |
| Day +100 | Not isolated | Not isolated |
| CD4+ (cells/mL) | ||
| Pre | 262 | 287 |
| Day +14 | 63 | |
| Day +28 | 21 | |
| Day +56 | 78 | 315 |
| Day +100 | 74 | 248 |
| Day +180 | 56 | NA |
| CD8 (cells/mL) | ||
| Pre | 391 | 381 |
| Day +14 | 23 | |
| Day +28 | 5 | |
| Day +56 | 26 | 176 |
| Day +100 | 141 | 128 |
| Day +180 | 379 | NA |
| CD3+ % donor | ||
| Day +28 | 51% | 48% |
| Day +56 | 69% | 48% |
| Day +100 | 95% | 49% |
| Day +180 | 100% | 61% |
| CD33+ % donor | ||
| Day +28 | 94% | 100% |
| Day +56 | 99% | 100% |
| Day +100 | 100% | 100% |
| Day +180 | 100% | 100% |
AML indicates acute myelogenous leukemia; B-B, beclamethasone + budesnonide; BO, bronchiolitis obliterans; CMV R/D, cytomegalovirus serology of recipient/donor; CSP, cyclosporine; Dx, diagnosis; FK, FK506; GVHD, graft-versus-host disease; HIV-ID, HIV-associated; HLA, human leukocyte antigen; MMF, mycophenolate mofetil; MSD, HLA-matched sibling donor; MUD, HLA-matched unrelated donor; NA, not applicable; and Pred, prednisone.
HIV monitoring
Plasma HIV RNA levels, peripheral blood mononuclear cell (PBMC) HIV proviral DNA levels, and PBMC quantitative HIV cultures were obtained at least weekly.4–6
IFN-γ ELISpot assays
Peptides based on optimal EBV- and CMV-derived HLA class I–restricted epitopes,7 obtained from Mabtech (Mariemont, OH) were tested individually and in pools. The ELISpot assay has been described.8 A positive response was defined as a 2-fold or greater increase in the mean number of spot-forming cells (SFCs) of experimental wells compared with negative controls, provided the mean SFC/106 cells in experimental wells was more than 50 after subtraction of negative controls.
CCR5 genotyping
HIV-1 coreceptor CCR5Δ32 genotype was determined using DNA restriction fragment length polymorphism analysis as previously described.8,9
Results and discussion
Feasibility of HAART administration and affect on viral replication during NMHCT
The choice of HAART took into consideration potency, adverse effects, and potential drug interactions. Patent 1 was switched to efavirenz to provide a more effective regimen and to avoid possible nevirapine-mediated hepatotoxicity associated with immune reconstitution.10 Protease inhibitors were not used, to avoid drug interactions causing toxic levels of CSP and the azoles.1 Although efavirenz induces P450 enzymes, we reasoned that the dosage of affected drugs could be adjusted upward. The risk of abacavir hypersensitivity was extremely unlikely, since neither patients nor donors expressed HLA-B*5701.11
After transplantation, full donor CD33+ chimerism was established in both patients. The CD3+ subset of patient 1 converted from 51% donor at day +28 to 100% donor by day +80 after withdrawal of immune suppression for treatment of recurrent leukemia, detected at day +42. In patient 2, the proportion of donor CD3+ cells continues to increase. Reconstitution of CD4+ and CD8+ subsets (Table 1) was typical for NMHCT recipients, half of whom receive prolonged immunosuppressive therapy.2 Patient 2 has not experienced opportunistic infections or GVHD and remains alive more than 180 days after HCT. Patient 1 developed CMV reactivation on day +83, successfully treated with foscarnet. GVHD grade 3 developed on day +56, after sudden withdrawal of immune suppression to treat recurrent leukemia, and responded to additional immune suppression. On day +100, patient 1 was discharged home on MMF, tacrolimus, and tapering doses of prednisone. Seven months after HCT he developed severe bronchiolitis obliterans (BO) unresponsive to additional immune suppression. He died 3 months later of pulmonary failure related to BO, Pseudomonas aeruginosa sepsis, and mucormycosis of his sinuses while on multiple immune suppressive agents.
For both patients at all time points after HCT the plasma HIV RNA remained undetectable and no HIV was detected by viral cultures of PBMC (Table 1). In patient 1, PBMC proviral DNA was detected at baseline before HCT and at day +28, but thereafter became undetectable when he converted to 100% donor chimerism. In contrast, proviral DNA was detected at all time points evaluated in patient 2, who continued to have mixed donor-host chimerism of the T-cell subset. Both patients' donor cells expressed wild-type CCR5 coreceptor, not the CCR5Δ32 allele, which is associated with resistance to HIV infection.12
These data show that HAART can be administered throughout NMHCT, including during conditioning, with control of plasma viral replication. Neither patient developed HIV-related complications, and, although our first patient did not survive, his death was due to transplantation-related complications of GVHD. In our experience before the advent of HAART, myeloablative HCT resulted in acceleration of HIV disease, persistent culturable virus, and high levels of HIV p24 antigen after transplantation.13 Other studies also demonstrated that myeloablative therapy alone did not eliminate viral reservoirs.14–19 In 2002, Kang et al reported on 2 patients given NMHCT with gene-modified sibling donor PBSC.20 HAART was discontinued 1 week before, then resumed after HCT. One patient developed an acute retroviral syndrome, with a 6-log rise in HIV load, which declined rapidly after reinstitution of HAART. He subsequently developed central nervous system toxoplasmosis and died of progressive Hodgkin lymphoma. The second patient developed no HIV-related complications and was alive at the time of report. Detailed immunologic studies were not reported.
Development of donor-derived antigen-specific T-cell responses
To determine whether HIV-naive donor cells could target HIV-epitope specificities, we examined T-cell responses to multiple optimal epitopes restricted by the patients' HLA class I alleles (Table 2). PBMCs obtained from patient 1 before transplantation reacted with 6 of 26 epitopes tested that spanned 6 proteins, including Nef, Vpr, Pol, Env, Gag, and Tat. Response frequencies to individual epitopes ranged from 100 to 805 SFC/106 PBMCs with a total response of 1545 SFCs/106 PBMCs. HIV-1–specific CD8+ T-cell responses were not detected on day +56, but were detected on day +81. The total magnitude of response frequency was 664 SFCs/106 PBMCs with response being targeted to 8 of 26 epitopes examined. The pattern of HIV-epitope specificities of posttransplantation donor cells was markedly different compared with pretransplantation recipient T cells. The inter-pretation of HIV-specific CD8 T-cell responses in patient 2, who has mixed donor-host CD3+ chimerism, is more complicated. Before transplantation, PBMCs reacted with 4 epitopes limited to Nef and Gag. After transplantation, 2 of these same epitopes were recognized, probably by residual host CD8 cells. In addition, T-cell responses were elicited by 2 new epitopes in RT and 1 new epitope involving Env.
Table 2.
Interferon-γ ELISpot responses before and after allogeneic hematopoietic cell transplantation
| Virus | Patient 1 |
Patient 2 |
||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| HLA restriction | Protein and region | Peptide sequence | IFN-γ SFC* |
HLA restriction | Protein and region† | Peptide sequence | IFN-γ SFC* |
|||
| Pre-HCT | Day +81 | Pre-HCT | Day +81 | |||||||
| CMV† | Pool | 678 | 487 | Pool | 0 | ND | ||||
| EBV† | Pool 1 | 435 | 1180 | Pool 1 | 3017 | 980 | ||||
| Pool 2 | 1870 | 137 | Pool 2 | 150 | 110 | |||||
| Pool 3 | 1013 | 620 | Pool 3 | 171 | 130 | |||||
| A2 | BMLFI (259-267) | GLCTLVAML | 120 | 120 | B8 | EBNA3A (158– 166) | QAKWRLQTL | 150 | ND | |
| A24 | BRLFI (28-37) | DYCNVLNKEF | 955 | 503 | BZLF1 (190–197) | RAKFKQLL | 3492 | 1110 | ||
| B27 | BRLFI (28-37) | RRIYDLIEL | 1455 | 237 | B35 | EBNA3A (458– 466) | YPLHEQHGM | 158 | 150 | |
| FluA† | A2 | Matrix 1 (58-66) | GILGFVFTL | 205 | 77 | B8 | NP (380–388) | ELRSRYWAI | 117 | − |
| B27 | NP (383-391) | SRYWAIRTR | 53 | 350 | ||||||
| HIV | A2 | Gag p17 (77-85) | SLYNTVATL | − | 67 | A26 | Gag p24 (35-43) | EVIPMFSAL | 142 | − |
| Env gp160 (311-320) | RGPGRAFVTI | 120 | 90 | B8 | Gag p17 (24-32) | GGKKKYKL | 129 | 50 | ||
| RT(33-41) | ALVEICTEM | − | 120 | Gag p24 (128-135) | EIYKRWII | 54 | − | |||
| Pol (22-31) | MASDFNLPPV | 195 | 63 | Nef (90-97) | FLKEKGGL | 158 | 50 | |||
| Vpr (59-67) | AIIRILQQL | 155 | − | RT (118-127) | VPLDEDFRKY | − | 100 | |||
| Nef (136-145) | PLTFGWCYKL | 170 | − | RT (18-26) | GPKVKQWPL | − | 90 | |||
| A24 | Gag p17 (28-36) | KYKLKHIVW | − | 53 | Env gp41 (95-103) | TAVPWNASW | − | 50 | ||
| B27 | Gag p24 (131-140) | KRWIILGLNK | 805 | − | ||||||
| Env gp160 (786-795) | GRRGWEALKY | − | 257 | |||||||
| Nef (105-114) | RRQDILDLWI | − | 77 | |||||||
| Cw12 | Tat (32-37) | CCFHCQVC | 100 | 67 | ||||||
CMV indicates cytomegalovirus; EBV, Epstein-Barr virus; HIV, human immunodeficiency virus; IFN, interferon; PBMC, peripheral blood mononuclear cell; and SFC, spot-forming cell.
The value of SFC/106 PBMC is shown for all positive responses. Negative responses are indicated by −. Patient 1 had no response in either the pre- or posttransplant samples to the following epitopes: HLA-A2–restricted: Env gp160 (813-822) SLLNATDIAV, RT (179-187) VIYQYMDDL, RT (309-317) ILKEPVHGV, Nef (180-189) VLEWRFDSRL; HLA-A2, A24 restricted: Nef (190-198) AFHHVAREL; HLA-A24–restricted: Gag p24 (162-172) RDYVDRFFKTL, Env gp160 (52-61) LFCASDAKAY, Env gp160 (585-593) RYLKDQQLL, Gag p17 (19-27) IRLRPGGKK; HLA-B39–restricted: Gag p24 (61-69) GHQAAMQML, HLA-Cw1–restricted: Gag p24 (36-43) VIPMFSAL, Gag_Pol_TF (24-31) NSPTRREL, Env gp160 (217-226) YCAPAGFAIL, Env gp160 (712-720) YSPLSLQTL, Vpu (74-82) HAPWDVNDL. Patient 2 had no response in either the pre or posttransplant samples to the following epitopes: HLA-B8–restricted: Gag p17 (74-81) ELRSLYNTV, Gag p24 (197-205) DCKTILKAL, Env gp120 (2-10) RVKEKYQHL, Env gp41 (75-82) YLKDQQLL, Nef (13-20) WPTVRERM, Env gp41 (337-345) LQGLERALL; HLA-B35–restricted: Gag p17 (36-44) WASRELERF, Gag p17 (124-132) NSSKVSQNY, Gag p24 (122-130) PPIPVGDIY, RT (107-115) TVLDVGDAY, RT (175-183) NPDIVIYQY, RT (175-183) HPDIVIYQY, Env gp120 (42-52) VPVWKEATTTL, Env gp120 (78-86) DPNPQEVVL, Nef (74-81) VPLRPMT.
T-cell responses to EBV, CMV, and FluA epitopes were used as controls. The CMV and EBV peptide pools consisted of published HLA class I–restricted CD8+ T-cell epitopes.
Allogeneic HCT provides a novel platform to study the development of HIV-1–specific T-cell responses generated from HIV-1–naive donor cells in the setting of chronic controlled infection. We found that new HIV-1–specific CD8+ T-cell responses were generated early after HCT, despite the absence of plasma HIV-1 RNA, suggesting that plasma viremia is not necessary for the development of an HIV-1 T-cell response and that T-cells can be primed by HIV antigens expressed in lymphatic tissue with limited HIV replication. The observed shift in recognized epitopes demonstrates that naive donor cells are capable of generating de novo HIV-1–specific immune responses under these conditions. Allogeneic HCT also provides a platform to study the effects of conditioning and the establishment of a new immune system, including antigen-presenting and -responding cells, on the latent HIV-1 reservoir. The gradual loss of detectable proviral DNA after HCT in patient 1, who achieved full donor chimerism, suggests that the pool of latently infected lymphocytes declined after HCT and that the priming of HIV-1–naive T-cells occurred with limited and localized HIV replication and antigen expression. These findings may provide insights for development of HIV-1 vaccines or immune-based therapies.
Acknowledgments
The authors thank Deborah Sessions for assistance in preparation of the manuscript.
The study was supported by grants CA78902, CA18029, CA15704, AI64061, AI57005, and K08 AI 059173 from the National Institutes of Health (Bethesda, MD).
Footnotes
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 USC section 1734.
Authorship
Contribution: A.W. conceived and designed the study and wrote the manuscript; U.M. designed and performed research assays and analyzed data; R.H. wrote the manuscript; J.M. performed research assays and analyzed data; T.M. designed and performed research assays and analyzed data; S.R. provided analytical agents and analyzed data; R.C. designed and performed research assays and analyzed data; F.A. provided resources and critical review of the manuscript; L.C. designed and performed research assays and analyzed data; and R.S. provided resources and supervised the study.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Ann Woolfrey, MD, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue N, Seattle, WA 98115; e-mail: awoolfre@fhcrc.org.
References
- 1.Krishnan A, Molina A, Zaia J, et al. Durable remissions with autologous stem cell transplantation for high-risk HIV-associated lymphomas. Blood. 2005;105:874–878. doi: 10.1182/blood-2004-04-1532. [DOI] [PubMed] [Google Scholar]
- 2.Maris MB, Niederwieser D, Sandmaier BM, et al. HLA-matched unrelated donor hematopoietic cell transplantation after nonmyeloablative conditioning for patients with hematologic malignancies. Blood. 2003;102:2021–2030. doi: 10.1182/blood-2003-02-0482. [DOI] [PubMed] [Google Scholar]
- 3.Maris MB, Sandmaier BM, Storer BE, et al. Unrelated donor granulocyte colony-stimulating factor-mobilized peripheral blood mononuclear cell transplantation after nonmyeloablative conditioning: the effect of postgrafting mycophenolate mofetil dosing. Biol Blood Marrow Transplant. 2006;12:454–465. doi: 10.1016/j.bbmt.2005.12.030. [DOI] [PubMed] [Google Scholar]
- 4.Zuckerman RA, Lucchetti A, Whittington WL, et al. Herpes simplex virus (HSV) suppression with valacyclovir reduces rectal and blood plasma HIV-1 levels in HIV-1/HSV-2-seropositive men: a randomized, double-blind, placebo-controlled crossover trial. J Infect Dis. 2007;196:1500–1508. doi: 10.1086/522523. [DOI] [PubMed] [Google Scholar]
- 5.Jackson JB, Drew J, Lin HJ, et al. Establishment of a quality assurance program for human immunodeficiency virus type 1 DNA polymerase chain reaction assays by the AIDS Clinical Trials Group. J Clin Microbiol. 1993;31:3123–3128. doi: 10.1128/jcm.31.12.3123-3128.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Jackson JB, Coombs RW, Sannerud K, Rhame FS, Balfour HH., Jr Rapid and sensitive viral culture method for human immunodeficiency virus type 1. J Clin Microbiol. 1988;26:1416–1418. doi: 10.1128/jcm.26.7.1416-1418.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Bihl F, Narayan M, Chisholm JV, III, et al. Lytic and latent antigens of the human gammaherpesviruses Kaposi's sarcoma-associated herpesvirus and Epstein-Barr virus induce T-cell responses with similar functional properties and memory phenotypes. J Virol. 2007;81:4904–4908. doi: 10.1128/JVI.02509-06. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Larsson M, Jin X, Ramratnam B, et al. A recombinant vaccinia virus based ELISPOT assay detects high frequencies of Pol-specific CD8 T cells in HIV-1-positive individuals. AIDS. 1999;13:767–777. doi: 10.1097/00002030-199905070-00005. [DOI] [PubMed] [Google Scholar]
- 9.Quillent C, Oberlin E, Braun J, et al. HIV-1-resistance phenotype conferred by combination of two separate inherited mutations of CCR5 gene. Lancet. 1998;351:14–18. doi: 10.1016/S0140-6736(97)09185-X. [DOI] [PubMed] [Google Scholar]
- 10.Baylor MS, Johann-Liang R. Hepatotoxicity associated with nevirapine use (Review). J Acquir Immune Defic Syndr. 2004;35:538–539. doi: 10.1097/00126334-200404150-00014. [DOI] [PubMed] [Google Scholar]
- 11.Phillips E, Mallal S. Drug hypersensitivity in HIV [review]. Curr Opin Allergy Clin Immunol. 2007;7:324–330. doi: 10.1097/ACI.0b013e32825ea68a. [DOI] [PubMed] [Google Scholar]
- 12.Huang Y, Paxton WA, Wolinsky SM, et al. The role of a mutant CCR5 allele in HIV-1 transmission and disease progression. Nat Med. 1996;2:1240–1243. doi: 10.1038/nm1196-1240. [DOI] [PubMed] [Google Scholar]
- 13.Bowden RA, Coombs RW, Nikora BH, et al. Progression of human immunodeficiency virus type-1 infection after allogeneic marrow transplantation. Am J Med. 1990;88:49N–52N. [PubMed] [Google Scholar]
- 14.Aboulafia DM, Mitsuyasu RT, Miles SA. Syngeneic bone-marrow transplantation and failure to eradicate HIV. AIDS. 1991;5:344. [PubMed] [Google Scholar]
- 15.Gabarre J, Leblond V, Sutton L, et al. Autologous bone marrow transplantation in relapsed HIV-related non-Hodgkin's lymphoma. Bone Marrow Transplant. 1996;18:1195–1197. [PubMed] [Google Scholar]
- 16.Davis KC, Hayward A, Ozturk G, Kohler PF. Lymphocyte transfusion in case of acquired immunodeficiency syndrome. Lancet. 1983;1:599–600. doi: 10.1016/s0140-6736(83)92855-6. [DOI] [PubMed] [Google Scholar]
- 17.Lane HC, Zunich KM, Wilson W, et al. Syngeneic bone marrow transplantation and adoptive transfer of peripheral blood lymphocytes combined with zidovudine in human immunodeficiency virus (HIV) infection. Ann Intern Med. 1990;113:512–519. doi: 10.7326/0003-4819-113-7-512. [DOI] [PubMed] [Google Scholar]
- 18.Vilmer E, Rhodes-Feuillette A, Rabian C, et al. Clinical and immunological restoration in patients with AIDS after marrow transplantation, using lymphocyte transfusions from the marrow donor. Transplantation. 1987;44:25–29. doi: 10.1097/00007890-198707000-00007. [DOI] [PubMed] [Google Scholar]
- 19.Verdonck LF, de Gast GC, Lange JM, Schuurman HJ, Dekker AW, Bast BJ. Syngeneic leukocytes together with suramin failed to improve immunodeficiency in a case of transfusion-associated AIDS after syngeneic bone marrow transplantation. Blood. 1988;71:666–671. [PubMed] [Google Scholar]
- 20.Kang EM, De Witte M, Malech H, et al. Nonmyeloablative conditioning followed by transplantation of genetically modified HLA-matched peripheral blood progenitor cells for hematologic malignancies in patients with acquired immunodeficiency syndrome. Blood. 2002;99:698–701. doi: 10.1182/blood.v99.2.698. [DOI] [PubMed] [Google Scholar]