John A. Zaia

A Randomized, Controlled Trial of Prophylactic Ganciclovir for Cytomegalovirus Pulmonary Infection in Recipients of Allogeneic Bone Marrow Transplants

Abstract Background. Cytomegalovirus (CMV)-associated interstitial pneumonia is a major cause of death after allogeneic bone marrow transplantation. We conducted a controlled trial of ganciclovir in recipients of bone marrow transplants who had asymptomatic pulmonary CMV infection. We also sought to identify risk factors for the development of CMV interstitial pneumonia. Methods. After bone marrow transplantation, 104 patients who had no evidence of respiratory disease underwent routine bronchoalveolar lavage on day 35. The 40 patients who had positive cultures for CMV were randomly assigned to either prophylactic ganciclovir or observation alone. Ganciclovir (5 mg per kilogram of body weight intravenously) was given twice daily for two weeks and then five times per week until day 120. Results. Of the 20 culture-positive patients who received prophylactic ganciclovir, 5 (25 percent) died or had CMV pneumonia before day 120, as compared with 14 of the 20 culture-positive control patients (70 percent) who w...

Towards an HIV cure: a global scientific strategy

Given the limitations of antiretroviral therapy and recent advances in our understanding of HIV persistence during effective treatment, there is a growing recognition that a cure for HIV infection is both needed and feasible. The International AIDS Society convened a group of international experts to develop a scientific strategy for research towards an HIV cure. Several priorities for basic, translational and clinical research were identified. This Opinion article summarizes the groups recommended key goals for the international community.

Cytomegalovirus in hematopoietic stem cell transplant recipients: current status, known challenges, and future strategies

Cytomegalovirus (CMV) infection is a major cause of morbidity and mortality after hematopoietic stem cell transplantation. Significant progress has been made in the prevention of CMV disease over the past decade, but prevention of late CMV disease continues to be a challenge in selected high-risk populations. The pretransplantation CMV serostatus of the donor and/or recipient remains an important risk factor for posttransplantation outcome despite the use of antiviral prophylaxis and preemptive therapy; CMV-seropositive recipients of T cell-depleted grafts in particular continue to have a survival disadvantage compared with seronegative recipients with seronegative donors. The risk of developing antiviral drug resistance remains low in most patients; however, in a setting of intense immunosuppression (eg, after transplantation from a haploidentical donor), the incidence may be as high as 8%. Primary CMV infection via blood transfusion can be reduced by the provision of seronegative or leukocyte-depleted blood products; however, a small risk of 1% to 2% of CMV disease remains. Surveillance and preemptive therapy are effective in preventing the sequelae of transfusion-related CMV infection. Indirect immunomodulatory effects of CMV are increasingly recognized in hematopoietic stem cell transplant recipients. Strategies currently being investigated include long-term suppression of CMV with valganciclovir for the prevention of late CMV infection and disease, adoptive transfer of CMV-specific T cells, and donor and recipient vaccination strategies.

Acyclovir Halts Progression of Herpes Zoster in Immunocompromised Patients

We conducted a placebo-controlled, double-blind study of acyclovir therapy for acute herpes zoster in immunocompromised patients. Of the 94 patients enrolled in the study, 52 had localized skin lesions at entry, and 42 had disseminated cutaneous zoster. A one-week course of intravenous acyclovir (1500 mg per square meter of body-surface area per day) halted progression of zoster in both groups, as determined by development or progression of cutaneous dissemination, development of visceral zoster, or proportion of cases deemed treatment failures. Significantly fewer patients treated with acyclovir within the first three days after the onset of exanthem had complications of zoster, as compared with patients treated with placebo (P = 0.02 by Fishers exact test), but acyclovir also stopped progression of zoster in patients treated after three days of rash (P = 0.05 by Fishers exact test). Acyclovir recipients with disseminated cutaneous zoster had a significantly accelerated rate of clearance of virus from vesicles, as compared with placebo recipients (P = 0.05 by the Breslow test).

RNA-Based Gene Therapy for HIV with Lentiviral Vector–Modified CD34+ Cells in Patients Undergoing Transplantation for AIDS-Related Lymphoma

Transfected stem cells transplanted into patients with HIV infection resulted in sustained RNA expression of introduced genes in blood cells for up to 2 years. Steps Toward a Stable Source of Therapeutic RNA Gene therapy in humans has not been easy to implement. Genes inserted into complex human cells have triggered serious unintended consequences and have often proven to be short-lived. Yet perseverance may be paying off. DiGiusto et al. report a step toward workable gene therapy in the form of stable expression of a lentiviral vector encoding anti-HIV RNAs in blood stem cells transplanted into AIDS patients. None of these patients is cured, but the vector seems to stably express the potentially therapeutic RNAs. Putting exogenous gene sequences into humans is risky, and review boards are appropriately conservative. But DiGiusto et al. took advantage of a clinical situation to design a trial that minimized extra risk to the subjects. Blood cancer (lymphoma) is common in AIDS patients, and they are often treated by ablation of their diseased bone marrow with chemotherapy followed by a transplant with their own previously saved blood stem cells. Because these patients were being transplanted with their own blood cells anyway, the authors were able to get permission to transfect a few of the blood cells of four patients with a vector carrying anti-HIV entities and reinfuse them along with the normally transplanted cells. The vector made RNAs that could counteract viral replication in several ways: inhibition of viral entry (with a CCR5 ribozyme), inhibition of RNA transport [by a small interfering RNA (siRNA) to tat/rev], and inhibition of viral transcription initiation with a decoy RNA. The good news was that the patients showed no signs of toxicity besides problems usually associated with transplantation and that blood cells from all four patients contained signs of the transplanted genes, with the amounts increasing in two of the patients after 18 months. Although the fraction of cells containing the genes was <0.2%, this was not too different from the fraction of transfected cells that was infused into the patients. The three anti-HIV RNAs could also be detected as long as 1 year after the initial infusion, and examination of T cells, monocytes, and B cells from one patient confirmed the presence of vector in these three cell types. These cells that survived for long periods of time in patients, although too scarce to cure or even improve their HIV infections, nevertheless offer lessons for future applications of gene therapy. We know that this procedure is seemingly safe and that cells given new genetic material via a lentiviral vector outside the patient can survive once reimplanted. Continued perseverance can only bring us closer to realizing the potential of this promising therapy. AIDS patients who develop lymphoma are often treated with transplanted hematopoietic progenitor cells. As a first step in developing a hematopoietic cell–based gene therapy treatment, four patients undergoing treatment with these transplanted cells were also given gene-modified peripheral blood–derived (CD34+) hematopoietic progenitor cells expressing three RNA-based anti-HIV moieties (tat/rev short hairpin RNA, TAR decoy, and CCR5 ribozyme). In vitro analysis of these gene-modified cells showed no differences in their hematopoietic potential compared with nontransduced cells. In vitro estimates of successful expression of the anti-HIV moieties were initially as high as 22% but declined to ~1% over 4 weeks of culture. Ethical study design required that patients be transplanted with both gene-modified and unmanipulated hematopoietic progenitor cells obtained from the patient by apheresis. Transfected cells were successfully engrafted in all four infused patients by day 11, and there were no unexpected infusion-related toxicities. Persistent vector expression in multiple cell lineages was observed at low levels for up to 24 months, as was expression of the introduced small interfering RNA and ribozyme. Therefore, we have demonstrated stable vector expression in human blood cells after transplantation of autologous gene-modified hematopoietic progenitor cells. These results support the development of an RNA-based cell therapy platform for HIV.

Novel Dual Inhibitory Function Aptamer–siRNA Delivery System for HIV-1 Therapy

The successful use of small interfering RNAs (siRNAs) for therapeutic purposes requires safe and efficient delivery to specific cells and tissues. In this study, we demonstrate cell type-specific delivery of anti-human immunodeficiency virus (anti-HIV) siRNAs through fusion to an anti-gp120 aptamer. The envelope glycoprotein is expressed on the surface of HIV-1-infected cells, allowing binding and internalization of the aptamer-siRNA chimeric molecules. We demonstrate that the anti-gp120 aptamer-siRNA chimera is specifically taken up by cells expressing HIV-1 gp120, and that the appended siRNA is processed by Dicer; this releases an anti-tat/rev siRNA which, in turn, inhibits HIV replication. We show for the first time a dual functioning aptamer-siRNA chimera in which both the aptamer and the siRNA portions have potent anti-HIV activities. We also show that gp120 expressed on the surface of HIV-infected cells can be used for aptamer-mediated delivery of anti-HIV siRNAs.

Prevention of Cytomegalovirus Infection by Cytomegalovirus Immune Globulin After Marrow Transplantation

In an effort to prevent cytomegalovirus infection among seronegative patients having marrow transplants, a globulin with high antibody levels against cytomegalovirus was given before and for 11 weeks after transplantation in a randomized trial. Among 36 patients who received no prophylactic granulocyte transfusions, globulin recipients had significantly fewer infections than controls (2 of 17 versus 8 of 19, p = 0.05 by Fishers exact test and p = 0.03 by Mantel-Cox test). Conversely, infection rates were high and unchanged by globulin use among patients who received granulocytes from seropositive donors (7 of 8 recipients versus 6 of 7 controls). The lack of effect of the globulin among patients receiving transfusions of granulocytes from seropositive donors may suggest that the dose of antibody was insufficient or that antibody is ineffective against virus transmitted in granulocytes. We conclude that cytomegalovirus infection can be prevented by immunoprophylaxis in seronegative patients having marrow transplants who are not given granulocyte transfusions.

Growth Inhibition by Acycloguanosine of Herpesviruses Isolated from Human Infections

Inhibition by acycloguanosine (ACG) of plaque formation by harpes simplex virus types 1 and 2 (HSV-1 and HSV-2), varicella-zoster virus, and cytomegalovirus was studied. Seventeen clinical isolates of HSV-1 were inhibited by ACG at a mean 50% inhibitory dose (ID50) of 0.15 ± 0.09 μM. The mean ID50 for 10 isolates of HSV-2 was 1.62 ± 0.76 μM, and for four isolates of varicella-zoster virus it was 3.75 ± 1.30 μM. The ID50s for two cytomegalovirus isolates were 100 and 160 μM, and for four additional isolates of cytomegalovirus no end point (ID50) was reached at 200 μM. ACG at a concentration of 200 μM had no effect on deoxyribonucleic acid synthesis in human fibroblast cells and only inhibited thymidine incorporation by Vero cells by one-third. These studies demonstrated the antiviral activity of ACG against clinical isolates of HSV-1, HSV-2, and varicella-zoster virus and the lack of toxicity to monkey or human cells in culture at concentrations which markedly inhibited these viruses. ACG had little effect on cytomegalovirus at concentrations in excess of 100 μM.

Airborne Transmission of Chickenpox in a Hospital

The occurrence of chickenpox in pediatric hospitals disrupts routine care of immunologically normal patients and is potentially life-threatening to immunosuppressed patients.1 , 2 Guidelines for pr...

Inhibition of HIV-1 infection by lentiviral vectors expressing Pol III-promoted anti-HIV RNAs.

A primary advantage of lentiviral vectors is their ability to pass through the nuclear envelope into the cell nucleus thereby allowing transduction of nondividing cells. Using HIV-based lentiviral vectors, we delivered an anti-CCR5 ribozyme (CCR5RZ), a nucleolar localizing TAR RNA decoy, or Pol III-expressed siRNA genes into cultured and primary cells. The CCR5RZ is driven by the adenoviral VA1 Pol III promoter, while the human U6 snRNA Pol III-transcribed TAR decoy is embedded in a U16 snoRNA (designated U16TAR), and the siRNAs were expressed from the human U6 Pol III promoter. The transduction efficiencies of these vectors ranged from 96-98% in 293 cells to 15-20% in primary PBMCs. A combination of the CCR5RZ and U16TAR decoy in a single vector backbone gave enhanced protection against HIV-1 challenge in a selective survival assay in both primary T cells and CD34(+)-derived monocytes. The lentiviral vector backbone-expressed siRNAs also showed potent inhibition of p24 expression in PBMCs challenged with HIV-1. Overall our results demonstrate that the lentiviral-based vectors can efficiently deliver single constructs as well as combinations of Pol III therapeutic expression units into primary hematopoietic cells for anti-HIV gene therapy and hold promise for stem or T-cell-based gene therapy for HIV-1 infection.