Allogeneic haematopoietic stem cell transplantation in patients with human immunodeficiency virus: the experiences of more than 25 years.

For treatment of several malignancies, transplantation of allogeneic haematopoietic stem cells (HSCT) derived from bone marrow or peripheral blood has been used as a therapeutic procedure for decades. In the past, HSCT has been suggested as a treatment option for infection with the human immunodeficiency virus type 1 (HIV-1), but these attempts were mostly unsuccessful. Today, after the introduction of an active anti-retroviral therapy, the lifetime expectancy of HIV-infected patients has improved substantially, but nevertheless the incidence rate of malignancies in these patients has increased considerably. Therefore, it can be assumed that there will be a rising necessity for HIV-1-infected patients with malignancies for allogeneic HSCT. At the same time, there is increasing interest in treatment methods which might target the HIV-1 reservoir more effectively, and the question has been raised as to whether allogeneic HSCT could be linked to such strategies. In this paper the data of more than 25 years experience with allogeneic HSCT in patients with HIV-1 are reviewed and analysed.

A fusion protein of HCMV IE1 exon4 and IE2 exon5 stimulates potent cellular immunity in an MVA vaccine vector.

A therapeutic CMV vaccine incorporating an antigenic repertoire capable of eliciting a cellular immune response has yet to be successfully implemented for patients who already have acquired an infection. To address this problem, we have developed a vaccine candidate derived from modified vaccinia Ankara (MVA) that expresses three immunodominant antigens (pp65, IE1, IE2) from CMV. The novelty of this vaccine is the fusion of two adjacent exons from the immediate-early region of CMV, their successful expression in MVA, and robust immunogenicity in both primary and memory response models. Evaluation of the immunogenicity of the viral vaccine in mouse models shows that it can stimulate primary immunity against all three antigens in both the CD4(+) and CD8(+) T cell subsets. Evaluation of human PBMC from healthy CMV-positive donors or patients within 6 months of receiving hematopoietic cell transplant shows robust stimulation of existing CMV-specific CD4(+) and CD8(+) T cell subsets.

Should HIV-positive patients with lymphoma be offered stem cell transplants?

Advances in effective antiretroviral therapy for HIV infection have made high-dose therapy and autologous stem cell transplantation possible in patients with HIV-associated lymphomas. Regimen-related toxicity is not significantly increased when antiretroviral therapy is combined with high-dose chemoradiotherapy. Durable engraftment can be seen with autologous stem cell rescue. Infectious complications can be managed with a combination of surveillance and prophylaxis. Long-term remissions of these high-risk lymphomas can be achieved with this approach. This suggests that patients with HIV-associated lymphomas should be considered for autologous transplantation in a manner similar to HIV-negative lymphoma patients.

Prevention and management of CMV-related problems after hematopoietic stem cell transplantation.

Prevention and management of human cytomegalovirus (CMV) infection after hematopoietic stem cell transplantation has improved substantially in the past decade. However, with this improvement, there is increased complexity in deciding which diagnostic tests, treatment strategies and immunologic assessments are optimal for different patient populations. The purpose of this review is to address certain practical problems that commonly arise and suggest a suitable approach to management that should have wide applicability.

Kinase-deficient CMVpp65 triggers a CMVpp65 specific T-cell immune response in HLA-A*0201.Kb transgenic mice after DNA immunization.

CMVpp65, a candidate component of human cytomegalovirus (CMV) vaccines, has phosphokinase (PK) activity that could affect vaccine safety. A mutated form of CMVpp65 substituting asparagine for lysine at the adenosine triphosphate (ATP)-binding site (CMVpp65mII) is kinase-deficient. Using DNA immunizations in a transgenic human leucocyte antigen (HLA)A*0201.Kb mouse model, the mutated CMVpp65 induced cytotoxic T lymphocytes (CTL) immunity similarly to native CMVpp65. Murine CTL lines generated from these immunizations killed human cells either after sensitization with CMVpp65-specific peptides or after infection with either CMV-Towne strain or rvac-pp65. It is proposed that CMVpp65mII be evaluated in candidate vaccines for CMV.

Autologous stem cell transplantation for HIV-associated lymphoma.

Is peripheral stem cell mobilization followed by autologous stem cell transplantation (ASCT) feasible in patients with human immunodeficiency virus (HIV)- associated lymphoma (HIV-L)? Studies have demonstrated that, in the HIV- negative (HIV(-)) setting, ASCT may improve lymphoma-free survival in high-risk non-Hodgkin lymphoma (NHL) or relapsed Hodgkin disease (HD) and NHL. Given the poor prognosis of HIV-L with conventional chemotherapy, this dose-intensive approach was explored. Nine patients with HIV-HD or NHL mobilized a median of 10.6 x 10(6) CD34(+) cells/kg and engrafted after ASCT. CD4 counts recovered to pretransplantation levels and HIV viral loads were controlled in patients compliant with antiretroviral therapy. Seven of 9 patients remain in remission from their lymphoma at a median of 19 months after transplantation. Thus, patients with HIV-L on antiretroviral therapy can engraft following ASCT. Prolonged lymphoma remissions, without significant compromise of immune function, can be seen, suggesting that ASCT can be used in selected patients with HIV-L.

Hematologic Aspects of HIV/AIDS.

This review addresses various aspects of HIV infection pertinent to hematology, including the consequences of HIV infection on specific aspects of hematopoiesis and an update on the current biologic, epidemiologic and therapeutic aspects of AIDS-related lymphoma and Hodgkin's disease. The results of the expanding use of progenitor cell transplantation in HIV infected patients are also reviewed. In Section I, Dr. Scadden reviews the basis for HIV dysregulation of blood cell production, focusing on the role of the stem cell in HIV disease. T cell production and thymic function are discussed, with emphasis placed upon the mechanisms of immune restoration in HIV infected individuals. Results of clinical and correlative laboratory studies are presented. In Section II, Dr. Levine reviews the recent epidemiologic trends in the incidence of lymphoma, since the widespread availability of highly active anti-retroviral therapy (HAART). The biologic aspects of AIDS-lymphoma and Hodgkin's disease are discussed in terms of pathogenesis of disease. Various treatment options for these disorders and the role of concomitant anti-retroviral and chemotherapeutic intervention are addressed. Drs. Zaia and Krishnan will review the area of stem cell transplantation in patients with AIDS related lymphoma, presenting updated information on clinical results of this procedure. Additionally, they report on the use of gene therapy, with peripheral blood CD34+ cells genetically modified using a murine retrovirus, as a means to treat underlying HIV infection. Results of gene transfer experiments and subsequent gene marking in HIV infected patients are reviewed.

The SH-3 and SH-4 antibodies recognize distinct epitopes on CD73 from human mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are pluripotent cells in the bone marrow that have the capacity to differentiate along a number of connective tissue lineages, including cartilage, bone, adipose tissue, and stroma. The SH-3 and SH-4 monoclonal antibodies recognize epitopes present on the surface of human MSCs. This study describes the isolation and characterization of the antigen that is recognized by these antibodies. A protein of molecular weight approximately 67 kDa was immunoprecipitated from a solubilized membrane preparation of human MSCs using the SH-3 antibody. Analysis of peptides derived from this protein by mass spectrometry and sequencing identified it as CD73 (ecto-5'-nucleotidase). The SH-4 antibody was also shown to react with purified bovine CD73 by immunoblotting, but the SH-3 antibody failed to react with the bovine protein. These results indicate that both SH-3 and SH-4 epitopes are present on CD73, but they are distinct. CD73, present in lymphoid tissue, plays a role in the activation of B-lymphocytes and in signal transduction in the hematopoietic compartment of bone marrow. The role that CD73 may play in bone marrow stromal interactions and in the differentiation of MSCs is discussed.

Human immunodeficiency virus-infected patients receiving highly active antiretroviral therapy maintain activated CD8+ T cell subsets as a strong adaptive immune response to cytomegalovirus.

CD8(+) T lymphocyte function specific for human cytomegalovirus (CMV) was evaluated in 14 patients infected with human immunodeficiency virus (HIV) receiving highly active antiretroviral therapy (HAART) and 26 CMV-seropositive donors without HIV infection. Fifty-seven percent of the HIV-infected group had CMV-specific cytolytic activity in freshly isolated peripheral blood mononuclear cells (PBMC) against targets expressing CMV pp65. Both interferon (IFN)-gamma secretion by CD8(+) T cells and the frequency of human leukocyte antigen (HLA)-tetramer-positive T cells in HLA-A*0201-positive HIV-infected subjects correlated with CMV-specific cytolysis. In contrast, PBMC from healthy CMV-seropositive donors did not have either measurable CMV-specific cytolysis or secretion of IFN-gamma without in vitro stimulation. The T helper response to CMV antigens was vigorous in healthy CMV-seropositive donors but low in the cohort of HIV-infected patients. Potent CD8(+) cytotoxic T lymphocyte responses to CMV in HIV-infected patients receiving HAART is the converse of what is found in healthy CMV-seropositive subjects and may be the predominant adaptive immune response against CMV in HIV-infected patients.

Randomized, placebo-controlled, double-blind study of a cytomegalovirus-specific monoclonal antibody (MSL-109) for prevention of cytomegalovirus infection after allogeneic hematopoietic stem cell transplantation.

MSL-109 is a monoclonal antibody specific to the cytomegalovirus (CMV) glycoprotein H with high neutralizing capacity. In a prospective, randomized, double-blind study, allogeneic hematopoietic stem cell transplantation (HSCT) recipients with positive donor and/or recipient serology for CMV before transplantation received either 60 mg/kg MSL-109 (n = 59), 15 mg/kg MSL-109 (n = 60), or placebo (n = 60) intravenously every 2 weeks from day -1 until day 84 after transplantation. CMV pp65 antigenemia, CMV-DNA load in plasma, and viremia by culture were tested weekly. Primary end points were development of pp65 antigenemia at any level and/or viremia for which ganciclovir was given. There was no statistically significant difference in CMV pp65 antigenemia or viremia among patients in the 60-mg group (pp65 antigenemia, 47%; viremia, 15%), the 15-mg group (52%; 23%), and the placebo group (45%; 17%). There was also no difference in maximum levels of pp65 antigenemia, time to clearance of pp65 antigenemia after start of ganciclovir, CMV disease, invasive bacterial and fungal infections, time to neutrophil and platelet engraftment, acute graft-versus-host disease, days of hospitalization, and overall survival rate among the 3 groups. However, a subgroup analysis of CMV-seronegative recipients with a seropositive donor (D+/R-) showed a transiently improved survival rate by day 100 in MSL-109 recipients (mortality: 60-mg group, 1/13; 15-mg group, 1/12; placebo group, 6/10 [P = .02 for 60-mg versus placebo groups; P = .08 for 15-mg versus placebo groups]); by the end of follow-up, the difference was no longer statistically significant. The improved survival rate in D+/R- patients could not be attributed to a reduction in CMV disease; however, MSL-109 was associated with improved platelet engraftment and less grade III to IV acute graft-versus-host disease in this subgroup. In a subgroup analysis of CMV-seropositive recipients of MSL-109 (D+/R+ and D-/R+), overall mortality was increased compared to that of the placebo group (P = .12 for the 60-mg versus placebo groups, P = .05 for the 15-mg versus placebo groups, and P = .04 for the dose levels combined versus placebo). MSL-109 was well tolerated and no immune response to the drug was observed. Thus, MSL-109 was safe but did not reduce CMV infection in allogeneic HSCT recipients. The transient survival advantage seen early after transplantation in CMV D+/R- patients and the negative effect on survival in seropositive patients remain unexplained. Thus, there is no evidence that MSL-109 is beneficial in CMV-seropositive HSCT recipients.

Site-directed mutation in a conserved kinase domain of human cytomegalovirus-pp65 with preservation of cytotoxic T lymphocyte targeting.

The major target of human cytomegalovirus (CMV)-specific cytotoxic T lymphocytes (CTL) is the tegument protein CMVpp65. However, this protein has protein kinase (PK) activity, and the unknown effects on cell replication of an exogenous PK in healthy cells could limit the use of CMVpp65 as a vaccine, especially in children. In this report we show that a point mutation converting lysine to asparagine at the invariant lysine (K436), an essential site for phosphotransfer, abolishes the threonine kinase activity. The mutant CMVpp65 maintains its immunologic target characteristics, including antibody and CTL reactivity. This kinase-deficient CMVpp65 is a candidate for evaluation in future CMV vaccine development.

Prospects for gene therapy using HIV-based vectors.

Recombinant vectors derived from murine leukemia virus (MLV) have been widely used to introduce genes in human gene therapy clinical trials and have shown the potential for medical applications and the promise of significantly improving medical therapies. Yet, the demonstrated limitations of these vectors support the need for continued development of improved vectors. The intrinsic properties associated with the MLV genome and its life cycle do not favor the successful application of this vector system in certain human gene transfer applications. Since MLV integrates randomly into the host genome, transgene expression is frequently affected by the flanking host chromatin. MLV insertions can often result in silencing or position effect variation of gene expression either immediately after insertion or following cell expansion in culture or in vivo. Migration of the MLV pre-integration complex from the cytoplasm into the nucleus of infected cells requires mitosis for nuclear membrane breakdown. Since a majority of human cells exist in a quiescent state in vivo, it is unlikely that direct in vivo gene delivery into target tissues can be achieved with the MLV vector system. Finally, insertion of tissue-specific cis-regulatory sequences to direct transgene expression frequently results in either the rearrangement of the vector sequence or disruption of the cis-regulatory sequence functions. The long terminal repeat (LTR) of MLV, which contains a ubiquitously active enhancer/promoter element, may partially account for this problem. Together, these problems pose a major obstacle for the use of MLV vectors in the treatment of human diseases. This Chapter discusses some of the potential targets to which HIV vectors might be applied in clinical settings and some of the issues surrounding use of HIV vectors in gene transfer clinical trials.

Alternative O-glycosylation/O-phosphorylation of the murine estrogen receptor beta.

Estrogen receptor beta, a homologue to estrogen receptor alpha, is a new member of the steroid hormone receptor family. Recently, we documented that estrogen receptor alpha, like other transcription factors, is modified by O-linked N-acetylglucosamine (O-GlcNAc), a ubiquitous transitory posttranslational modification on nuclear and cytoplasmic proteins. Here, we report that estrogen receptor beta is alternatively modified by either O-GlcNAc or O-phosphate. Lectin chromatography of in vitro translated protein first suggested that murine estrogen receptor beta (mER-beta) is O-GlcNAcylated. Structural characterization of the carbohydrate moieties on mER-beta, overexpressed in insect Sf9 cells, confirmed the presence of O-GlcNAc. mER-beta, overexpressed in mammalian cells, is also O-GlcNAcylated. The major site of O-GlcNAc on mER-beta from Sf9 cells is Ser(16) near the N-terminus. Concomitant analyses also documented the O-phosphorylation of mER-beta at Ser(16). MALDI-TOF mass spectrometry showed alternative occupancy of this locus by these two abundant and dynamic posttranslational modifications. The localization of a major O-GlcNAc/O-phosphate site in proximity of the transactivation domain and as part of a PEST region (target sequences for rapid protein degradation) on mER-beta suggests that these modifications may play a role in regulating estrogen receptor beta transactivation and turnover.

High dose therapy and autologous stem cell transplantation for human immunodeficiency virus-associated non-Hodgkin lymphoma in the era of highly active antiretroviral therapy.

BACKGROUND: The advent of highly active antiretroviral therapy (HAART) has allowed the exploration of more dose-intensive therapy such as autologous stem cell transplantation (ASCT) in selected patients with human immunodeficiency virus (HIV)-associated non-Hodgkin lymphoma (NHL). METHODS: The authors report on the use of myeloablative chemotherapy with ASCT in two HIV positive patients with NHL. The first patient underwent ASCT at the time of first disease remission for poor risk, diffuse, large cell NHL and the second patient had multiply recurrent, chemosensitive Burkitt lymphoma. ASCT was performed in both patients using a transplant conditioning regimen of high dose cyclophosphamide, carmustine, and etoposide (CBV). RESULTS: The target dose of >/= 5 x 10(6)/kg CD34 positive peripheral blood stem cells (PBSC) utilized for ASCT was collected using granulocyte-colony stimulating factor (G-CSF) after chemotherapy for mobilization while both patients were receiving concomitant HAART for HIV infection. HAART was continued during CBV conditioning. Prompt hematopoietic recovery was observed after ASCT. Both patients remained in clinical disease remission from their lymphoma at 28 months and 20 months after transplant, respectively. CONCLUSIONS: ASCT is feasible in patients with HIV-associated NHL. Adequate numbers of CD34 positive PBSC can be procured from patients receiving HAART and chemotherapy for NHL. Selected patients with HIV-related lymphoma can tolerate the high dose CBV myeloablative chemotherapy regimen without increased acute regimen-related toxicity. Reinfusion of G-CSF-mobilized PBSC can lead to rapid recovery of hematologic function and sustained engraftment after ASCT. Given the poor prognosis of patients with HIV-associated NHL treated with conventional chemotherapy, further investigation of this approach should be considered.

The monoclonal antibody SH-2, raised against human mesenchymal stem cells, recognizes an epitope on endoglin (CD105).

Mesenchymal stem cells are multipotent cells resident in the bone marrow throughout adulthood which have the capacity to differentiate into cartilage, bone, fat, muscle, and tendon. A number of monoclonal antibodies raised against human MSCs have been shown to react with surface antigens on these cells in vitro. A protein of molecular mass 92 kDa was immunoprecipitated using the SH-2 monoclonal antibody. This was purified and identified by peptide sequencing analysis and mass spectrometry as endoglin (CD105), the TGF-beta receptor III present on endothelial cells, syncytiotrophoblasts, macrophages, and connective tissue stromal cells. Endoglin on MSCs potentially plays a role in TGF-beta signalling in the control of chondrogenic differentiation of MSCs and also in mediating interactions between MSCs and haematopoietic cells in the bone marrow microenvironment.

Comparison of amphotropic and pseudotyped VSV-G retroviral transduction in human CD34+ peripheral blood progenitor cells from adult donors with HIV-1 infection or cancer.

In this study we compared the transduction efficiency of conventional amphotropic MoMLV (LPONL[A]) with the MoMLV pseudotyped with that of VSV-G (LPONL[G]) in peripheral blood progenitor cells (PBPCs) from cancer patients and human immunodeficiency virus (HIV)-infected donors. The results showed that LPONL(A) and LPONL(G) infected the progenitor cells from these sources with equal efficiencies. The transgene neoR was detectable by polymerase chain reaction assay in colonies from 14-day colony-forming unit (CFU) assays and in those derived from long-term culture-initiating cell (LTC-ICs) assays. Although the overall levels of transduction efficiency were similar in cord blood and PBPCs from noninfected cancer donors (25-22%) when either LPONL(G) or LPONL(A) was used, they were significantly lower in HIV-1-infected donors compared with noninfected cancer donors when LPONL(G) was used (13 vs. 25%; p = 0.027), and when LPONL(A) was used (12 vs. 22%; p = 0.087). The clonogenic potentials of infected and noninfected CD34+ cells were similar; thus no toxicity could be attributed to the virus preparation. We conclude that PBPCs from HIV-1-infected individuals are transduced less efficiently than those from non-HIV-infected cancer donors. Nonetheless, PBPCs from HIV-infected persons serve as potential targets in gene therapy for acquired immune deficiency syndrome.

A study of the pharmacokinetics, antiviral activity, and tolerability of oral ganciclovir for CMV prophylaxis in marrow transplantation.

Oral ganciclovir is effective in preventing cytomegalovirus (CMV) disease in HIV-infected patients despite a bioavailability of only 6-9%. To determine safety, pharmacokinetics, and the influence of acute gastrointestinal graft-vs.-host disease (GI-GVHD) on the bioavailability and antiviral effect of oral ganciclovir after marrow transplantation, CMV seropositive patients received oral ganciclovir (1000 mg 3 times per day) from day 35 (+/- 7 days) until day 100 after transplantation. Single-dose (intravenous and oral) and steady-state oral pharmacokinetic profiles and weekly trough levels were performed. Twenty-one patients received oral ganciclovir (seven with GI-GVHD, 14 without); 17 had steady-state pharmacokinetic profiles and seven had single-dose profiles. The absolute bioavailability was similar in patients with or without acute GI-GVHD (7.2 vs. 6.9%). At steady state, the extent and rate of absorption of oral ganciclovir were comparable in these same patient subgroups (area under the curve [AUC] = 13.5 and 10.2 mg x hours/L, respectively; time to peak serum ganciclovir concentrations = 5.5 and 3.8 hours, respectively). Breakthrough CMV antigenemia, viremia, or plasma polymerase chain reaction positivity occurred in eight of 21 (38%) patients (four of seven with GVHD and four of 14 without). Drug discontinuation because of GI adverse effects was required in six of 21 (29%) patients. Neutropenia occurred in two of 15 (13%) patients who had received oral ganciclovir for more than 10 days. In conclusion, the bioavailability of oral ganciclovir seems similar to that reported in other settings. The presence of acute GVHD of the GI tract did not appear to adversely affect absorption of oral ganciclovir. The use of oral ganciclovir was limited by the presence of GI intolerance in the early posttransplant period. The efficacy of oral ganciclovir in preventing CMV infection in marrow transplant recipients is being assessed in a separate randomized controlled trial.

Copurification of P6, MRP8, and MRP14 from human granulocytes and separation of individual proteins.

A method is described for purification of P6, MRP8, and MRP14, three calcium-binding proteins assigned to the S100 protein family. The purification procedure included preparation of human granulocytes, ammonium sulfate precipitation, and anion-exchange chromatography and resulted in the copurification of P6, MRP8, and MRP14. Individual proteins were separated by either preparative isoelectric focusing or preparative SDS-PAGE. The procedure was carried out in the course of 4 days and yielded several milligrams of essentially pure P6, MRP8, and MRP14 in either native or denatured form.

Virion encapsidation of tRNA(3Lys)-ribozyme chimeric RNAs inhibits HIV infection.

Retroviruses require a specific host cellular tRNA primer for initiation of first-strand DNA synthesis. This primer is bound by viral proteins and copackaged into virions. We have exploited this property in the design and testing of an antiviral ribozyme fused to tRNA(3Lys), the primer used for lentiviral replication, including human immunodeficiency virus (HIV-1 and HIV-2). The chimera consists of tRNA(3Lys) covalently attached to a hammerhead ribozyme, which is targeted to the region immediately upstream of the primer binding site of the HIV-1 genome. The tRNA-ribozyme chimeric transcript is catalytically active in vitro and is efficiently bound by HIV reverse transcriptase with an affinity similar to that of tRNA(3Lys). We have expressed the chimeric RNAs from either the tRNA(3Lys) intragenic RNA polymerase III promoter or from a human U6 snRNA promoter. The U6 promoter results in up to 10-fold enhanced expression of the tRNA-ribozyme. Most importantly, the tRNA(3Lys)-ribozymes are encapsidated in HIV-1 virions such that they are effective in substantially reducing the level of infectious virus produced from cells cotransfected with HIV-1 proviral DNA. These results demonstrate the feasibility of using this novel strategy to reduce HIV infectivity and more generally indicate the potential power of using the retroviral primer tRNAs as tools for expressing and delivering ribozymes and other antiretroviral RNAs to the virion capsid.