Pharmacologic control of homeostatic and antigen-driven proliferation to target HIV-1 persistence.

The presence of latent human immunodeficiency virus 1 (HIV-1) in quiescent memory CD4 + T cells represents a major barrier to viral eradication. Proliferation of memory CD4 + T cells is the primary mechanism that leads to persistence of the latent reservoir, despite effective antiretroviral therapy (ART). Memory CD4 + T cells are long-lived and can proliferate through two mechanisms: homeostatic proliferation via γc-cytokine stimulation or antigen-driven proliferation. Therefore, therapeutic modalities that perturb homeostatic and antigen-driven proliferation, combined with ART, represent promising strategies to reduce the latent reservoir. In this study, we investigated a library of FDA-approved oncology drugs to determine their ability to inhibit homeostatic and/or antigen-driven proliferation. We confirmed potential hits by evaluating their effects on proliferation in memory CD4 + T cells from people living with HIV-1 on ART (PLWH) and interrogated downstream signaling of γc-cytokine stimulation. We found that dasatinib and ponatinib, tyrosine kinase inhibitors, and trametinib, a MEK inhibitor, reduced both homeostatic and antigen-driven proliferationby >65%, with a reduction in viability <45%, ex vivo. In memory CD4 + T cells from PLWH, only dasatinib restricted both homeostatic and antigen-driven proliferation and prevented spontaneous rebound, consistent with promoting a smaller reservoir size. We show that dasatinib restricts IL-7 induced proliferation through STAT5 phosphorylation inhibition. Our results establish that the anti-cancer agent dasatinib is an exciting candidate to be used as an anti-proliferative drug in a clinical trial, since it efficiently blocks proliferation and iswell tolerated in patients with chronic myeloid leukemia (CML).

ATR and GADD45alpha mediate HIV-1 Vpr-induced apoptosis.

The human immunodeficiency virus type-1 (HIV-1) accessory gene vpr encodes a conserved 96-amino-acid protein that is necessary and sufficient for the HIV-1-induced block of cellular proliferation. Expression of vpr in CD4+ lymphocytes results in G2 arrest, followed by apoptosis. In a previous study, we identified the ataxia telangiectasia-mutated (ATM) and Rad3-related protein (ATR) as a cellular factor that mediates Vpr-induced cell cycle arrest. In the present study, we report that the breast cancer-associated protein-1 (BRCA1), a known target of ATR, is activated in the presence of Vpr. In addition, the gene encoding the growth arrest and DNA damage-45 protein alpha (GADD45alpha), a known transcriptional target of BRCA1, is upregulated by Vpr in an ATR-dependent manner. We demonstrate that RNAi-mediated silencing of either ATR or GADD45alpha leads to nearly complete suppression of the proapoptotic effect of Vpr. Our results support a model in which Vpr-induced apoptosis is mediated via ATR phosphorylation of BRCA1, and consequent upregulation of GADD45alpha.

Development of an Rev-independent, minimal simian immunodeficiency virus-derived vector system.

Lentiviral vectors are attractive candidates for gene therapy because of their ability to integrate into nondividing cells. To date, conventional HIV-1-based vectors can be produced at higher titers, but concerns regarding their safety for human use exist because of the possibility of recombination leading to production of infectious virions with pathogenic potential. Development of lentivirus vectors based on nonhuman lentiviruses constitutes an active area of research. We described a novel HIV-SIV hybrid vector system in which an HIV-1-derived transfer vector is encapsidated by SIVmac1A11 core particles and pseudotyped with VSV glycoprotein G. In an effort to further develop this vector system, we modified the packaging plasmid by deletion of the SIV accessory genes. Specifically, versions of the packaging plasmid (SIVpack) lacking vif, vpr, vpx, and/or nef were constructed. Our results indicate that, as with HIV-1-based packaging plasmids, deletion of accessory genes has no significant effect on transduction in either dividing or nondividing cells. The SIV packaging plasmid was also modified with regard to the requirement for RRE and rev. Deletion of the RRE and rev from SIVpack led to dramatic loss of transduction ability. Introduction of the 5' LTR from the spleen necrosis virus to packaging plasmids lacking RRE/Rev was then sufficient to fully restore vector titer. A minimal SIV transfer vector was also developed, which does not require RRE/Rev and exhibits no reduction in transduction efficiency in two packaging systems. The SIV-based vector system described here recapitulates the biological properties of minimal HIV-1-derived systems and is expected to provide an added level of safety for human gene transfer. We suggest that the SIV-derived vector system will also be useful to deliver anti-HIV-1 gene therapy reagents that would inhibit an HIV-1-derived vector.

Human T-cell leukemia viruses: epidemiology, biology, and pathogenesis.

The human T-cell lymphotropic viruses type I and type II are closely related human retroviruses that have similar biological properties, genetic organization and tropism for T lymphocytes. Along with the simian T-cell lymphoma virus type I, they define the group of retroviruses known as the primate T-cell leukemia/lymphoma viruses. Initially identified in 1980, the human T-cell lymphotropic virus type I has been implicated as the etiologic agent of adult T-cell leukemia/lymphoma and of a degenerative neurologic disorder known as tropical spastic paraparesis or human T-cell lymphotropic virus type I-associated myelopathy. The intriguing link between human T-cell lymphotropic virus type, T-cell malignancy, and a totally unrelated and non-overlapping neurological disorder suggests divergent and unique pathogenetic mechanisms. This review will address the epidemiology, molecular biology, and pathogenesis of human T-cell leukemia viruses.

Lentiviral vectors and gene therapy.

Gene therapy is a novel method under investigation for the treatment of genetic, metabolic and neurologic diseases, cancer and AIDS. The primary goal of gene therapy is to deliver a specific gene to a pre-determined target cell, and to direct expression of such a gene in a manner which will result in a therapeutic effect. Retroviral vectors have the ability to integrate in the host cell DNA irreversibly and therefore, are suitable vectors for permanent genetic modification of cells. Retrovirus-mediated gene transfer has been limited, however, by the inability of onco-retroviruses to productively infect non-dividing cells. Lentiviruses are unique among retroviruses because of their ability to infect target cells independently of their proliferation status. This chapter presents an up-to-date description of available lentiviral vectors, including vector design, applications to disease treatment and safety considerations. In addition, general aspects of the biology of lentiviruses with relevance to vector development will be discussed.

Inhibition of HIV type 1 infection with a RANTES-IgG3 fusion protein.

The natural ligands for the chemokine receptors CCR5 (RANTES, MIP-1alpha, and MIP-1beta) and CXCR4 (SDF-1) can act as potent inhibitors of infection by the human immunodeficiency virus type 1 (HIV-1) at the level of viral entry. Unlike antibody-mediated inhibition, chemokine-mediated inhibition is broadly effective. Different HIV-1 strains can utilize the same coreceptor(s) for viral entry and, therefore, can be blocked by the same chemokine(s). HIV-1 strains that are highly resistant to neutralization by V3-specific antibodies are sensitive to inhibition by chemokines. Therefore, the use of chemokine-derived molecules constitutes a potential therapeutic approach to prevent infection by HIV-1. We have generated a fusion protein between RANTES and human IgG3 (RANTES-IgG3). The effectiveness of RANTES-IgG3 inhibition of infection by HIV-1 was similar to that of rRANTES. Inhibition of HIV-1 by RANTES-IgG3 was specific for CCR5-dependent but not CXCR4-dependent HIV-1 isolates. Fusion of a chemokine to an IgG moiety offers two desirable properties with respect to the recombinant chemokine alone. First, IgG fusion proteins have extended half-lives in vivo. Second, molecules with IgG heavy chain moieties may be able to cross the placenta and potentially induce fetal protection.

Primary isolate neutralization by HIV type 1-infected patient sera in the era of highly active antiretroviral therapy.

Sera from highly selected HIV-1-positive patients are known to have the ability to neutralize a diverse array of primary isolates of HIV-1. The human osteosarcoma cell line that expresses CD4 and chemokine receptors (GHOST cells) was adapted to study HIV-1 neutralization in 37 HIV-1-infected individuals who were selected because of slow disease progression or nonprogression. Many of these individuals were receiving combination drug therapy. Molecularly cloned HIV-1 JR-FL and NL4-3 viruses were used as prototypes to define assay conditions. Sera were then tested at a 1:40 dilution against six additional primary isolates, three of which utilized CCR5 and three of which used both CCR5 and CXCR4. The assay was highly reproducible and independent of viral input titer, with a readout at 48 hr equivalent to that at later time points. As previously reported, neutralization sensitivity was entirely independent of coreceptor usage. Only a few sera from slow progressors were able to neutralize a broad array of primary isolates at a 1:40 dilution, and the best clinical predictor of broadly neutralizing antibody for primary isolates was the present use of antiretroviral agents. In further studies it was found that purified antibody accounted for the majority of the measured neutralization. However, experiments with exogenous addition of antiviral agents showed that the use of nucleosides also greatly contributed to the measured neutralization in some patients. Measurement of neutralization of HIV-1 primary isolates by sera from patients receiving antiretroviral therapy must be carried out with some caution.

HTLV type 1 Tax transduction in microglial cells and astrocytes by lentiviral vectors.

Infection with human T cell leukemia virus type 1 (HTLV-1) can result in the development of HAM/TSP, a nonfatal, chronic inflammatory disease involving neuronal degeneration and demyelination of the central nervous system. Elevated levels of the proinflammatory cytokines tumor necrosis factor alpha (TNF-alpha), interleukin-6 (IL-6), and IL-1 observed in the cerebrospinal fluid of HAM-TSP patients suggest that cytokine dysregulation within the CNS is involved in neuropathogenesis. HTLV-1 infection and enhanced expression of TNF-alpha by microglial cells, astrocytes, and macrophages has been hypothesized to lead to the destruction of myelin and oligodendrocytes in the CNS. Although the association of HTLV-2 infection and development of neurological disease is more tenuous, HTLV-2 has also been found to be associated with peripheral neuropathies. To investigate the roles of HTLV Tax(1) and Tax(2) in the induction of cytokine disregulation in these cell types, we are currently developing gene delivery vectors based on human immunodeficiency virus type-1 (HIV-1) capable of stably coexpressing the HTLV-1 or -2 tax and eGFP reporter genes in primary human cells. Transduction frequencies of up to 50%, as assessed by eGFP expression, can be achieved in human monocyte-derived macrophages and in explanted cultures of human microglia. Preliminary data suggest that Tax(1) expression is sufficient to up-regulate the proinflammatory cytokine profile in explanted human microglial cells. Future experiments will compare and evaluate the effect of tax(1) and tax(2) gene expression on the cellular proinflammatory cytokine expression profile, as well as demonstrate the effects of transducing human fetal astrocytes and PBMC-derived macrophages.

Genetic and biological comparisons of pathogenic and nonpathogenic molecular clones of simian immunodeficiency virus (SIVmac).

Simian immunodeficiency virus (SIV) is a designation for a group of related but unique lentiviruses identified in several primate species. A viral isolate from a rhesus macaque (i.e., SIVmac) causes a fatal AIDS-like disease in experimentally infected macaques, and several infectious molecular clones of this virus have been characterized. This report presents the complete nucleotide sequence of molecularly cloned SIVmac1A11, and comparisons are made with the sequence of molecularly cloned SIVmac239. SIVmac1A11 has delayed replication kinetics in lymphoid cells but replicates as well as uncloned SIVmac in macrophage cultures. Macaques infected with virus from the SIVmac1A11 clone develop antiviral antibodies, but virus does not persist in peripheral blood mononuclear cells and no disease signs are observed. SIVmac239 infects lymphoid cells, shows restricted replication in cultured macrophages, and establishes a persistent infection in animals that leads to a fatal AIDS-like disease. Both viruses are about 98% homologous at the nucleotide sequence level. In SIVmac1A11, the vpr gene as well as the transmembrane domain of env are prematurely truncated, whereas the nef gene of SIVmac239 is prematurely truncated. Sequence differences are also noted in variable region 1 (V1) in the surface domain of the env gene. The potential implications of these and other sequence differences are discussed with respect to the phenotypes of both viruses. This animal model is critically important for investigating the roles of specific viral genes in viral/host interactions that cannot be studied in individuals infected with the human immunodeficiency virus (HIV).

Functional and immunological characterization of SIV envelope glycoprotein produced in genetically engineered mammalian cells.

Retroviral envelope glycoproteins interact with cell receptors and are targets for antiviral immune responses in infected hosts. Macaque simian immunodeficiency virus (SIVmac) is a T-lymphocytopathic lentivirus which causes an AIDS-like disease in rhesus macaques. The envelope gene of SIVmac encodes a precursor glycoprotein (gp160) which is cleaved into an external domain (gp130) and a transmembrane domain (gp32). To investigate the functional and immunological properties of the SIV external envelope glycoprotein, we have used genetically engineered mammalian cells to produce recombinant gp130 (rgp130). The rgp130 has the appropriate molecular weight, is glycosylated, and has native conformation as determined by binding to the cell receptor for SIV, the CD4 antigen. Rhesus macaques immunized with purified rgp130 formulated in muramyl dipeptide adjuvant generated high titers of antienvelope antibodies. Antibodies from these macaques were tested for in vitro virus neutralization; very low or undetectable levels of neutralization were observed. In contrast, neutralizing antibodies were readily detected in sera from goats immunized with rgp130. With respect to cell-mediated immunity, proliferative responses to rgp130 were demonstrated in peripheral blood monocyte cells (PBMC) from macaques immunized with the recombinant glycoprotein as well as in PBMC from SIV-infected animals. These results show that rgp130 is functional and immunogenic; the potential of rgp130 for protective immunization remains to be determined.

Characterization of rhesus macaque B-lymphoblastoid cell lines infected with simian type D retrovirus.

A simian type D retrovirus designated SRV induces a fatal immunosuppressive disease in rhesus macaques. This syndrome shows many clinical similarities to acquired immunodeficiency syndrome (AIDS) in human immunodeficiency virus-infected individuals. To investigate the mechanisms of immune dysfunction in SRV infection, we have focused on the interactions of SRV serotype 1 (SRV-1) with macaque B-lymphoblastoid cell lines (B-LCL). Procedures were optimized for establishing B-LCL by immortalization of macaque B lymphocytes with rhesus Epstein-Barr virus (EBV). These cell lines express B-cell surface markers, secrete immunoglobulins of the IgG or IgM isotypes, and release EBV which transforms monkey B cells. In vitro cultures of B-LCL supported replication of SRV-1. Several B-LCL infected with SRV-1 showed downregulation of major histocompatibility complex (MHC) class II antigen expression whereas levels of MHC class I antigen remained unchanged. Infection of B-LCL with SRV-1 did not alter the level of secreted immunoglobulin. Rhesus EBV was also used to obtain B-LCL from macaques infected with SRV-1; these cell lines were found to release infectious SRV-1. Investigations on the interactions of SRV-1 with B cells will be useful for elucidating mechanisms involved in the immunopathogenesis of primate retroviruses.

Lentivirus and foamy virus vectors: novel gene therapy tools.

The aim of gene therapy is to modify the genetic material of living cells to achieve therapeutic benefit. Gene therapy involves the insertion of a functional gene into a cell, to replace an absent or defective gene, or to fight an infectious agent or a tumour. At present, a wide variety of somatic tissues are being explored for the introduction of foreign genes with a view towards treatment. A prime requirement for successful gene therapy is the sustained expression of the therapeutic gene without any adverse effect on the recipient. A highly desirable vector would be generated at high titres, integrate into target cells (including non-dividing cells) and have little or no associated immune reactions. Lentiviruses have the ability to infect dividing and non-dividing cells and, therefore, constitute ideal candidates for development of vectors for gene therapy. This review presents a description of available lentiviral vectors, including vector design, applications to disease treatment and safety considerations. In addition, general aspects of the biology of lentiviruses with relevance to vector development will be discussed. Recent investigations have revealed that foamy viruses, another group of retroviruses, are also capable of infecting non-dividing cells. Thus, foamy virus vectors are actively being developed in parallel to lentivirus vectors. This review will also include various aspects of the biology of foamy viruses with relevance to vector development.

Induction of cell-cycle arrest in cervical cancer cells by the human immunodeficiency virus type 1 viral protein R.

OBJECTIVE: To determine the ability of the human immunodeficiency virus type 1 (HIV-1) gene vpr to induce cell-cycle arrest in cervical cancer cells with or without human papillomavirus (HPV) type 16 E6 or E7 expression. METHODS: High- and low-level expression vectors for vpr (designated pVPR(HIGH) and pVPR(LOW), respectively) were used in conjunction with HPV-16 E6 or E7 vectors to transfect HPV-negative C33A cervical cancer cells. Vpr expression vectors encode a cell surface marker gene, murine Thy-1, for specific detection of transfected cells. Dual staining for the surface molecule Thy-1 and DNA content was used to determine cell-cycle profile and G2-phase arrest. RESULTS: C33A cells not expressing HPV-16 E6 showed some but not maximal G2-phase arrest when transfected with pVPR(HIGH) alone (43.2% of cells in the G2 phase). Addition of HPV-16 E6 or E6 plus E7 to pVPR(HIGH) substantially increased the percentages of cells in the G2 phase (51.3% and 53.0%, respectively). Cotransfection with pVPR(HIGH) and HPV-16 E7 did not increase significantly the percentage of cells in the G2 phase compared with pVPR(HIGH) alone (40.6% versus 43.2%). In transfections involving pVPR(LOW), a slight degree of G2-phase arrest was observed when Vpr was expressed alone (29.0% of cells in the G2 phase) or in cotransfection with HPV-16 E7 (33.2% of cells), and G2-phase arrest was augmented with the addition of HPV-16 E6 (41.7%) or E6 plus E7 (45.7%). CONCLUSION: Cervical cancer cells are susceptible to cell-cycle arrest induced by HIV-1 vpr. This effect is exacerbated by coexpression of HPV-16 E6, although E6 alone is incapable of inducing any detectable G2-phase arrest, suggesting that E6 and VPR share links in cell-cycle signaling pathways.

[Epidemiology and burden of acute otitis media in Valencia (Spain)]. / Epidemiología e impacto de la otitis media aguda en la Comunidad Valenciana.

OBJECTIVE: To assess the burden (incidence, treatment and complications) of acute otitis media (AOM) and otitis media with effusion (OME) in children younger than 5 years of age from Valencia, Spain. SUBJECT AND METHODS: We performed a retrospective cohort study of 1,399 children followed-up for the first 5 years of life. Seventeen pediatricians reviewed the medical records of their patients born in 1995 and 1996 and followed-up from birth until the age of 5 years. For each child, the number of otitis episodes, treatment, complications, and surgical interventions was obtained. RESULTS: There were 2,961 episodes of AOM in the first 5 years of life (2.23 cases/child). Four hundred seventy-six cases (16.1 %) occurred before 1 year of age and 1,346 between the first and second year of life (45.5 %). By the third year of life, 59.8 % had had at least one episode. In most children (80.9 %), diagnosis was made in primary care and required a median of 1.81 visits/episode for follow-up. A total of 94.5 % were treated with antibiotics (amoxicillin-clavulanate 38.8 %, cefuroxime 14.3 %, clarithromycin 8.2 % and amoxicillin 5.9 %) and 8.5 % required a change of antibiotic therapy. Two hundred seventeen children (15.2 %) had at least one episode of OME. Twenty-six patients (1.8 %, 95 % CI: 1.2-2.7 %) required insertion of ventilation tubes. Twenty-four patients (1.7 %) had secondary hypoacusis. There was one case of meningitis and two cases of chronic otorrhea. No cases of mastoiditis were recorded. CONCLUSIONS: The incidence of AOM in Valencia is 40,014 episodes/100,000 children younger than 5 years/year (95 % CI: 39,700-40,300). It represents a significant burden due to the large number of visits, antibiotic use, associated surgical procedures and need for auditory rehabilitation.

Apoptosis in AIDS.

Infection with the human immunodeficiency virus type 1 (HIV-1) leads to progressive immunodeficiency and onset of opportunistic infections and neoplasms. The loss of immune competence is associated with declines in both the functionality and the number of CD4+ lymphocytes. Multiple mechanisms have been proposed to explain death and dysfunction of CD4+ T-cells. The mechanisms of HIV-1-mediated cell death which are relevant in vivo are unclear at present. However, in vitro explorations on the cytopathic effects of HIV-1 have yielded a wealth of potential triggering events, and signaling and effector pathways leading to apoptosis. The types of pro- and anti-apoptotic stimuli that have been associated with HIV-1 are multiple and often appear overlapping or even contradictory. This review focuses on the various molecular determinants from HIV-1 that play a role in induction of apoptosis in T-lymphocytes. Special attention is devoted to the viral genes, env, nef, tat and vpr, for which a significant body of literature on apotosis-related effects is available.

Genetic analysis of human immunodeficiency virus type 1 integrase and the U3 att site: unusual phenotype of mutants in the zinc finger-like domain.

Retroviral integration is the step which leads to establishment of the provirus, cis- and trans-acting regions of the human immunodeficiency type 1 (HIV-1) retrovirus genome, including the attachment site (att) at the ends of the unintegrated viral DNA and the conserved domains within the integrase (IN) protein, have been identified as being important for integration. We investigated the role of each of these regions in the context of an infectious HIV-1 molecular clone through point mutagenesis of the att site and the zinc finger-like and catalytic domains of IN. The effect of each mutation on integration activity was examined by using a single-step infection system with envelope-pseudotype virus. The relative integration efficiency was estimated by monitoring the levels of viral DNA over time in the infected cells. The integration activities of catalytic domain point mutants and att site deletion mutants were estimated to be 0.5 and 5% of wild-type activity, respectively. However, in contrast with previous in vitro cell-free integration studies, alteration of the highly conserved CA dinucleotide resulted in a mutant which still retained 40% of wild-type integration activity. The relative levels of expression of each mutant, as measured by a luciferase reporter gene, correlated with levels of integration. This observation is consistent with those of previous studies indicating that integration is an obligatory step for retroviral gene expression. Interestingly, we found that three different HIV-1 constructs bearing point mutations in the zinc finger-like domain synthesized much lower levels of viral DNA after infection, suggesting impairment of these mutants before or at the initiation of reverse transcription. Western blot (immunoblot) analysis demonstrated wild-type levels of reverse transcriptase within the mutant virions. In vitro endogenous reverse transcription assays indicated that all three mutants in the zinc finger-like domain had wild-type levels of reverse transcriptase activity. These data indicate that in addition to integration, IN may have an effect on the proper course of events in the viral life cycle that precede integration.

Efficient gene transfer into human monocyte-derived macrophages using defective lentiviral vectors.

Gene therapy is a promising approach for the treatment of neurological disorders. However, current approaches to gene transfer in the central nervous system (CNS) are limited by the lack of effective, but non-invasive methods to deliver transgenes across the blood-brain barrier (BBB). In an effort to begin to explore the use of migratory monocytes as vehicles for delivery of therapeutic and antiviral genes into the CNS, we have utilized three HIV-based transfer vectors encoding cis-acting elements but lacking either structural genes (gag/pol and env), most accessory genes (vif, vpr and nef) and/or rev. These defective lentiviral vectors (DLV) encode the green fluorescent protein (GFP), display potent antiviral activity in CD4+ lymphocytes and can be mobilized by wild-type HIV-1 DLV were generated by transient transfection of 293T cells. Vector titers ranged from 4.2-6.6 x 10(6) infectious units (IU)/ml prior to concentration (by ultracentrifugation) and were equal to or higher than 1 x 10(9) IU/ml after concentration. Primary human monocyte-derived macrophages (MDM) were exposed to DLV resulting in efficiencies of transduction ranging from 14 to 26%. GFP expression in transduced MDM remained stable for more than 8 weeks without apparent cytopathic effect. Given the previously reported antiviral activities of these DLV and their lack of cytopathic effects on primary MDM, it may be possible to use these vectors to inhibit HIV-1 replication within the CNS.

Upregulation of survivin by HIV-1 Vpr.

The human survivin gene belongs to the family of inhibitor of apoptosis proteins (IAP) and is involved in apoptosis inhibition and regulation of cell division. The survivin gene is the only member of the IAP family whose expression is known to be regulated through the cell cycle. Survivin expression reaches the highest levels during the G(2)/M transition and then is rapidly degraded during the G(1) phase. Here we report that the human immunodeficiency virus type 1 (HIV-1) upregulates Survivin expression via survivin promoter transactivation. Vpr, an HIV-1 accessory protein that induces cell cycle arrest in G(2)/M, is necessary and sufficient for this effect. Blocking Vpr-induced G(2)/M arrest leads to elimination of the survivin promoter transactivation by Vpr. Our results suggest that Survivin may be actively involved in regulating cell viability during HIV-1 infection.