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.

Inhibition of HIV-1 replication with designed miRNAs expressed from RNA polymerase II promoters.

Small interfering RNA (siRNA) mediates sequence-specific RNA cleavage and represents a potential approach to treat the infection of human immunodeficiency virus (HIV). Expression of a single siRNA species frequently led to the emergence of HIV escape variants. Thus, multiple siRNAs targeted to different regions in the HIV-1 genome may be required. However, overexpression of different anti-HIV siRNA genes from multiple pol III promoters can induce cell toxicity, thus may not be a viable option in the setting of human gene therapy trials. In the current study, we evaluated the strategy of using pol II promoters to drive the expression of siRNAs against HIV-1. We replaced the stem sequence in the stem-loop structure of the well-characterized miR-30a with siRNA sequences and showed that designed microRNA (miRNA) could be expressed from pol II promoters. We demonstrated efficient inhibition of HIV-1 replication with such designed miRNA, but the efficacy was directly correlated with the expression level. Both the vector copy number and the promoter strength directly affected the ability of the siRNA to inhibit HIV-1 replication. We also showed that a combination of pol II and pol III promoters to express two different siRNAs increased the efficacy against HIV-1 replication without comprising cell viability.

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.

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.

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.

Infrequent occurrence of natural mutations in the pp65(495-503) epitope sequence presented by the HLA A*0201 allele among human cytomegalovirus isolates.

To determine if mutations of an immunodominant HLA-restricted cytomegalovirus (CMV) peptide sequence occur in nature, the sequence corresponding to the HLA A*0201-specific peptide CMVpp65(495-503) was determined in 50 human CMV isolates. Rare mutations were detected; 6 of 50 were silent mutations at the amino terminus of the peptide, while 3 of 50 were mutations of the native methionine residue to isoleucine (M499I). The observed M499I mutation in three isolates decreased cytolytic targeting.

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.

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.

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.

Late cytomegalovirus disease in marrow transplantation is predicted by virus load in plasma.

Late occurrence of cytomegalovirus (CMV) disease after day 100 after bone marrow transplantation has become an increasing problem; whether a quantitative measurement of CMV DNA in plasma by polymerase chain reaction (P-PCR) could be predictive of such disease was investigated. In a prospective study, 117 subjects undergoing allogeneic marrow transplantation were followed for 120 days with weekly CMV blood cultures, with day 35 bronchoalveolar lavage CMV cultures, with weekly CMV P-PCR, and with clinical follow-up for an additional 1-2 years. Despite preemptive ganciclovir, CMV disease occurred in 9% of subjects, with a median time of onset of 176 days. Quantitative CMV P-PCR was associated with the late development of CMV disease (P = .01). Of 43 subjects with positive P-PCR results, 23% developed CMV disease, but no disease occurred in the 74 subjects with negative P-PCR (P < .001), despite the fact that 22% had CMV isolated from lung lavage fluid and 32% had CMV isolated from blood.

Plasma polymerase chain reaction for cytomegalovirus DNA after allogeneic marrow transplantation: comparison with polymerase chain reaction using peripheral blood leukocytes, pp65 antigenemia, and viral culture.

In a prospective longitudinal study, detection of cytomegalovirus (CMV) DNA in plasma (plasma polymerase chain reaction [PCR]) was compared with PCR of CMV DNA in peripheral blood leukocytes (PBL PCR), the CMV pp65 antigenemia assay, and viral cultures from blood, urine, and throat of 29 patients, 14 of whom received pp65 antigenemia-guided early ganciclovir treatment and 15 of whom received ganciclovir at engraftment. Among 328 blood samples tested by all methods, PBL PCR was the most sensitive test, followed by the pp65 antigenemia assay, plasma PCR, and viremia. In the 14 patients who received pp65 antigenemia-guided early treatment, the incidence of PBL PCR, pp65 antigenemia, plasma PCR, and viremia before day 100 was 79%, 79%, 71%, and 27%, respectively, with a median day of onset of day 32, 42, 45, and 51, respectively. Nine patients (64%) became positive by PBL PCR, pp65 antigenemia, and plasma PCR. Of 15 patients who were treated with ganciclovir at engraftment, 12 (80%) became positive by PBL PCR, plasma PCR, and/or pp65 antigenemia while receiving ganciclovir; 3 (20%) had breakthrough infection with all three methods, including 2 with high-grade antigenemia (more than three positive cells in duplicate staining); none of these patients subsequently developed positive CMV cultures or disease. In 49 specimens, PBL PCR and/or pp65 antigenemia assay could not be performed because of insufficient neutrophil counts. In conclusion, the sensitivity of plasma PCR is significantly lower than that of PBL PCR but similar to that of the pp65 antigenemia assay. Plasma PCR may be particularly useful in clinical situations in which a less sensitive and possibly more specific assay is warranted or in which leukocyte counts are inadequate to perform cell-based assays.

Hematopoietic potential and retroviral transduction of CD34+ Thy-1+ peripheral blood stem cells from asymptomatic human immunodeficiency virus type-1-infected individuals mobilized with granulocyte colony-stimulating factor.

The potential of hematopoietic stem cells (HSCs) from human immunodeficiency virus type-1 (HIV-1)-infected individuals, eg, self-renewal and multilineage differentiative capacity, might be perturbed due to the underlying disease. In this study, we assessed the HSC activity in the CD34+ Thy-1+ cell population of peripheral blood stem cells (PBSCs) of three asymptomatic HIV-1-infected individuals after granulocyte colony-stimulating factor (G-CSF; 10 microg/kg/d) mobilization. On day 4 of G-CSF treatment, 0.8% to 1% of the total blood mononuclear cells were CD34+. Leukapheresis followed by a two-step cell isolation process yielded a CD34+ Thy-1+ cell population of high purity (76% to 92% CD34+ Thy-1+ cells). This cell population showed no evidence of HIV-1-containing cells based on a semiquantitative HIV-1 DNA polymerase chain reaction. Furthermore, the purified cells showed normal hematopoietic potential in in vitro clonogenic assays. Successful gene transfer into committed progenitor cells (colony-forming units-cells) and more primitive stem/progenitor cells (long-term culture colony-forming cells) could be shown after amphotropic retroviral transduction. These data provide evidence that the CD34+ Thy-1+ stem cell compartment can be mobilized and enriched in early stage HIV-1-infected patients. Furthermore, successful transduction of this cell population as a prerequisite for stem cell-based clinical gene therapy protocols was demonstrated.

Inhibition of human immunodeficiency virus-1 (HIV-1) replication after transduction of granulocyte colony-stimulating factor-mobilized CD34+ cells from HIV-1-infected donors using retroviral vectors containing anti-HIV-1 genes.

Transfer of "anti-HIV-1 genes" into hematopoietic stem cells of human immunodeficiency virus-1 (HIV-1)-infected individuals may be a potent therapeutic approach to render mature cells arising from transduced stem cells resistant to the destructive events associated with HIV-1 infection. To determine the feasibility of gene therapy for acquired immunodeficiency syndrome in individuals already infected with HIV-1, granulocyte colony-stimulating factor mobilized peripheral blood CD34+ cells were isolated from HIV-1-infected individuals and transduced with retroviral vectors containing three different anti-HIV-1-genes: the Rev binding domain of the Rev Responsive Element (RRE decoy) (L-RRE-neo), a double hammerhead ribozyme vector targeted to cleave the tat and rev transcripts (L-TR/TAT-neo), and the trans-dominant mutant of rev (M10) (L-M10-SN). As a control, a vector mediating only neomycin resistance (LN) was used. After 3 days of transduction on allogeneic stroma in the presence of stem cell factor, interleukin-6 (IL-6), and IL-3, the cultures were G418-selected, and then challenged with HIV-1(JR-FL) and a primary HIV-1 isolate. Compared with the control cultures, the L-RRE-neo-, L-TR/TAT-neo-, and L-M10-SN-transduced cultures displayed up to 1,000-fold inhibition of HIV-1 replication after challenge with HIV-1(JR-FL) and the primary HIV-1 isolate. Growth of the hematopoietic cells in long-term bone marrow culture was not perturbed by the presence of any of the anti-HIV-1 genes. This study shows that anti-HIV-1 genes can be introduced into CD34+ cells from individuals already infected with HIV-1, and strongly inhibit HIV-1 replication in primary monocytes derived from the CD34+ progenitors.

Evaluation of a quantitative plasma PCR plate assay for detecting cytomegalovirus infection in marrow transplant recipients.

A plasma PCR test, using a nonradioactive PCR plate assay, was evaluated for detection of human cytomegalovirus reactivation. This assay was compared to Southern blotting and found to perform well. As a noncompetitive method of quantitation, it was similar to a competitive method for detecting the number of genome copies per milliliter of plasma in marrow transplant recipients. This is a technically simplified assay with potential for adaptation to automation.