RESUMEN

Autologous and allogeneic haematopoietic stem cell (HSC) transplantation has been performed in patients with various malignant and non-malignant haematological disorders for more than 50 years. Ex vivo gene modification of HSCs for autologous transplantation opens up new therapeutic avenues for genetic and infectious diseases. Major advances have been made over the last three decades with respect to gene modification of HSCs and transplantation strategies, ultimately culminating in the approval of two such therapies in Europe (Strimvelis for a rare primary immune deficiency, and LentiGlobin for beta-thalassaemia). Newer gene-modifying technologies and treatment regimens have also recently come to the fore, which hold great promise for the development of safer and more effective treatments. We provide an overview of the current state of gene-modified HSC therapies, highlighting success stories, limitations and important considerations for achieving successful translation of these therapies to the clinic.

Asunto(s)

Terapia Genética/métodos , Enfermedades Hematológicas/terapia , Trasplante de Células Madre Hematopoyéticas/métodos , Células Madre Hematopoyéticas/citología , Humanos

RESUMEN

Autologous and allogeneic haematopoietic stem cell (HSC) transplantation has been performed in patients with various malignant and non-malignant haematological disorders for more than 50 years. Ex vivo gene modification of HSCs for autologous transplantation opens up new therapeutic avenues for genetic and infectious diseases. Major advances have been made over the last three decades with respect to gene modification of HSCs and transplantation strategies, ultimately culminating in the approval of two such therapies in Europe (Strimvelis for a rare primary immune deficiency, and LentiGlobin for beta-thalassaemia). Newer gene-modifying technologies and treatment regimens have also recently come to the fore, which hold great promise for the development of safer and more effective treatments. We provide an overview of the current state of gene-modified HSC therapies, highlighting success stories, limitations and important considerations for achieving successful translation of these therapies to the clinic

Asunto(s)

Servicios de Laboratorio Clínico , Células Madre Hematopoyéticas , Sistema Hematopoyético , Aplicaciones de la Informática Médica , Sudáfrica , Trasplante de Células Madre

RESUMEN

The persistence of latently HIV-infected cells in patients under combined antiretroviral treatment (cART) remains the major hurdle for HIV eradication. Thus far, individual compounds have not been sufficiently potent to reactivate latent virus and guarantee its elimination in vivo. Thus, we hypothesized that transcriptional enhancers, in concert with compounds triggering the innate immune system, are more efficient in reversing latency by creating a Th1 supportive milieu that acts against latently HIV-infected cells at various levels. To test our hypothesis, we screened six compounds on a coculture of latently infected cells (J-lat) and monocyte-derived dendritic cells (MDDCs). The protein kinase C (PKC) agonist prostratin, with a Toll-like receptor 8 (TLR8) agonist, resulted in greater reversion of HIV latency than any single compound. This combinatorial approach led to a drastic phenotypic and functional maturation of the MDDCs. Tumor necrosis factor (TNF) and cell-cell interactions were crucial for the greater reversion observed. Similarly, we found a greater potency of the combination of prostratin and TLR8 agonist in reversing HIV latency when applying it to primary cells of HIV-infected patients. Thus, we demonstrate here the synergistic interplay between TLR8-matured MDDCs and compounds acting directly on latently HIV-infected cells, targeting different mechanisms of latency, by triggering various signaling pathways. Moreover, TLR8 triggering may reverse exhaustion of HIV-specific cytotoxic T lymphocytes that might be essential for killing or constraining the latently infected cells. IMPORTANCE: Curing HIV is the Holy Grail. The so-called "shock and kill" strategy relies on drug-mediated reversion of HIV latency and the subsequent death of those cells under combined antiretroviral treatment. So far, no compound achieves efficient reversal of latency or eliminates this latent reservoir. The compounds may not target all of the latency mechanisms in all latently infected cells. Moreover, HIV-associated exhaustion of the immune system hinders the efficient elimination of the reactivated cells. In this study, we demonstrated synergistic latency reversion by combining agonists for protein kinase C and Toll-like receptor 8 in a coculture of latently infected cells with myeloid dendritic cells. The drug prostratin stimulates directly the transcriptional machinery of latently infected cells, and the TLR8 agonist acts indirectly by maturing dendritic cells. These findings highlight the importance of the immune system and its activation, in combination with direct-acting compounds, to reverse latency.

Asunto(s)

VIH-1/efectos de los fármacos , VIH-1/fisiología , Ésteres del Forbol/farmacología , Receptor Toll-Like 8/agonistas , Activación Viral/efectos de los fármacos , Adulto , Animales , Recuento de Linfocito CD4 , Comunicación Celular , Línea Celular , Técnicas de Cocultivo , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Femenino , Infecciones por VIH/inmunología , Infecciones por VIH/metabolismo , Infecciones por VIH/virología , Humanos , Masculino , Persona de Mediana Edad , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteína Quinasa C beta/metabolismo , Quinasa Syk/metabolismo , Subgrupos de Linfocitos T/metabolismo , Subgrupos de Linfocitos T/virología , Factor de Necrosis Tumoral alfa/metabolismo , Carga Viral , Latencia del Virus

RESUMEN

Endemic Burkitt's lymphoma (BL) is considered to preferentially develop in equatorial Africa because of chronic co-infection with Epstein-Barr virus (EBV) and the malaria pathogen Plasmodium falciparum. The interaction and contribution of both pathogens in the oncogenic process are poorly understood. Earlier, we showed that immune activation with a synthetic Toll-like receptor 9 (TLR9) ligand suppresses the initiation of EBV lytic replication in primary human B cells. In this study we investigate the mechanism involved in the suppression of EBV lytic gene expression in BL cell lines. We show that this suppression is dependent on functional TLR9 and MyD88 signaling but independent of downstream signaling elements, including phosphatidylinositol-3 kinase, mitogen-activated protein kinases and nuclear factor-kappaB. We identified TLR9 triggering resulting in histone modifications to negatively affect the activation of the promoter of EBV's master regulatory lytic gene BZLF1. Finally, we show that P. falciparum hemozoin, a natural TLR9 ligand, suppresses induction of EBV lytic gene expression in a dose-dependent manner. Thus, we provide evidence for a possible interaction between P. falciparum and EBV at the B-cell level and the mechanism involved in suppressing lytic and thereby reinforcing latent EBV that has unique oncogenic potential.

Asunto(s)

Linfoma de Burkitt/patología , Herpesvirus Humano 4/genética , Histonas/metabolismo , Receptor Toll-Like 9/metabolismo , Transactivadores/genética , Transcripción Genética , Animales , Linfocitos B/efectos de los fármacos , Linfocitos B/metabolismo , Linfocitos B/patología , Linfocitos B/virología , Secuencia de Bases , Linfoma de Burkitt/virología , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Islas de CpG/genética , Hemoproteínas/metabolismo , Hemoproteínas/farmacología , Herpesvirus Humano 4/efectos de los fármacos , Herpesvirus Humano 4/fisiología , Humanos , Ligandos , Factor 88 de Diferenciación Mieloide/genética , Plasmodium falciparum/metabolismo , Regiones Promotoras Genéticas/genética , Proteínas Protozoarias/metabolismo , Proteínas Protozoarias/farmacología , Transducción de Señal/efectos de los fármacos , Transcripción Genética/efectos de los fármacos , Activación Viral/efectos de los fármacos

RESUMEN

Advances in generation of mice that on human hematopoietic stem and progenitor cell transplantation develop and maintain human hemato-lymphoid cells have fueled an already thriving field of research. We focus here on human T cell development and HIV infection in Rag2 -/- gamma(c) -/- mice transplanted as newborns with human CD34+ cord blood hematopoietic stem and progenitor cells.

Asunto(s)

Modelos Animales de Enfermedad , Infecciones por VIH/inmunología , Hematopoyesis/fisiología , Linfocitos T/inmunología , Linfocitos T/virología , Animales , Proteínas de Unión al ADN/deficiencia , Trasplante de Células Madre Hematopoyéticas , Humanos , Subunidad gamma Común de Receptores de Interleucina/deficiencia , Ratones , Ratones SCID

Asunto(s)

Analgésicos Opioides/efectos adversos , Antirretrovirales/administración & dosificación , Infecciones por VIH/tratamiento farmacológico , Metadona/efectos adversos , Torsades de Pointes/inducido químicamente , Esquema de Medicación , Combinación de Medicamentos , Interacciones Farmacológicas , Quimioterapia Combinada , Femenino , Humanos , Lopinavir , Persona de Mediana Edad , Pirimidinonas/administración & dosificación , Ritonavir/administración & dosificación

RESUMEN

The HIV pandemic continues. Today the number of HIV-infected people is estimated to be 39.4 million. Despite huge efforts it is unlikely that there will be an efficient HIV vaccine available in the near future. Until now, only two phase III HIV vaccination trials have been performed in man. However, transmission could not be prevented. The hurdle for a rational approach to generate a vaccine is the still incomplete knowledge about HIV pathogenesis and the high rate of mutations of HIV. In this review we discuss the distinct aspects of the immune response, which could be exploited for HIV vaccine strategies and describe current vaccine approaches.

Asunto(s)

Vacunas contra el SIDA/administración & dosificación , Vacunas contra el SIDA/inmunología , Infecciones por VIH/inmunología , Infecciones por VIH/prevención & control , VIH-1/inmunología , Vacunación Masiva/métodos , Vacunación/métodos , Vacunación/tendencias , Humanos

RESUMEN

A 29-year-old man with rapidly destructive Staphylococcus epidermidis endocarditis after mitral valve reconstruction is presented. Resistance to rifampin and teicoplanin occurred during antibiotic treatment resulting in clinical failure and valve destruction. Subsequently, the patient was successfully treated, by combining valve replacement with antibiotic therapy including quinupristin/dalfopristin, levofloxacin, and vancomycin. In conclusion, S. epidermidis can cause rapid valve destruction with large vegetations, and combination of surgery and antibiotic therapy may be necessary.

Asunto(s)

Endocarditis Bacteriana/microbiología , Infecciones Estafilocócicas/diagnóstico , Staphylococcus epidermidis/aislamiento & purificación , Adulto , Antibacterianos/uso terapéutico , Progresión de la Enfermedad , Farmacorresistencia Bacteriana Múltiple , Endocarditis Bacteriana/tratamiento farmacológico , Endocarditis Bacteriana/cirugía , Humanos , Masculino , Infecciones Estafilocócicas/tratamiento farmacológico , Infecciones Estafilocócicas/microbiología , Staphylococcus epidermidis/efectos de los fármacos

RESUMEN

BACKGROUND: Varicella zoster virus (VZV) causes significant morbidity and mortality in immunocompromised patients. Subclinical reactivation has been described in solid organ recipients and has been associated with graft versus host disease in bone marrow transplantation. Newer studies assessing the prevalence and impact of subclinical VZV reactivation in solid organ transplant (SOT) recipients are lacking. METHODS AND RESULTS: In a first step we developed a highly sensitive quantitative polymerase chain reaction (qPCR) assay for VZV DNA with a detection limit of < or = 20 copies/mL. Using this assay, we retrospectively analyzed plasma samples of different patient groups for VZV DNA. VZV DNA was found in 10/10 plasma samples of immunocompetent patients with herpes zoster (VZV copy numbers/mL: mean+/-SEM 1710+/-1018), in 1/1 sample of a human immunodeficiency virus-infected patient with primary VZV disease (15,192 copies/mL) and in 4/4 plasma samples of immunocompromised patients with visceral VZV disease (mean of first value 214,214+/-178,572). All 108 plasma samples of asymptomatic SOT recipients off any antiviral therapy, randomly sampled over 1 year, were negative for VZV DNA. CONCLUSION: Our qPCR assay proved to be highly sensitive (100%) in symptomatic VZV disease. We did not detect subclinical reactivation in asymptomatic SOT recipients during the first post-transplant year. Thus, subclinical VZV reactivation is either a rare event or does not exist. These data need to be confirmed in larger prospective trials.

Asunto(s)

ADN Viral/sangre , Dosificación de Gen/genética , Herpesvirus Humano 3/aislamiento & purificación , Trasplante de Órganos/efectos adversos , Reacción en Cadena de la Polimerasa/métodos , Adulto , Varicela/inmunología , Varicela/virología , Herpes Zóster/inmunología , Herpes Zóster/virología , Herpesvirus Humano 3/genética , Humanos , Inmunocompetencia , Huésped Inmunocomprometido , Sensibilidad y Especificidad , Viremia/inmunología , Viremia/virología

RESUMEN

BACKGROUND: Patients with hematological malignancies are at increased risk for various infections. In patients with solid cancer, a variety of immunosuppressive mechanisms affecting T-cell response are described. We hypothesized that patients with advanced solid tumors may exhibit an impaired recognition of viral antigens. To test this, the capability of CD8+ T cells to recognize recall antigens from influenza and vaccinia virus was compared in patients and healthy individuals. Since all patients and most of the healthy individuals had been vaccinated against vaccinia years ago, comparison of the two groups was expected to be especially informative with respect to distinct effector T-cell reactivity. MATERIALS AND METHODS: Our test population included 16 healthy individuals and 12 patients with advanced solid cancers who were currently not receiving chemotherapy. We stimulated peripheral blood mononuclear cells (PBMC) ex vivo with the well-characterized influenza A matrix 58-66 peptide and the immunogenic and HLA-A*0201 restricted peptide epitope SLSAYIIRV derived from the modified vaccinia virus Ankara (MVA). A specific CD8+ T-cell reactivity was determined by quantitative real-time polymerase chain reaction (qRT-PCR) measuring changes in interferon gamma (IFN-gamma) mRNA expression levels. RESULTS: We found that significantly fewer cancer patients than healthy individuals exhibited specific T-cell recognition of the vaccinia epitope (25% and 69%, respectively). In addition, strength of the T-cell responses against both viral peptides was significantly reduced in cancer patients. CONCLUSION: Patients with advanced tumors are Less likely to mount a T-cell response against viral epitopes. These findings may have implications for the design of immunotherapeutic interventions against virus-induced diseases, including tumors.

Asunto(s)

Antígenos Virales/inmunología , Linfocitos T CD8-positivos/inmunología , Neoplasias/inmunología , Adulto , Femenino , Humanos , Virus de la Influenza A/inmunología , Interferón gamma/biosíntesis , Masculino , Persona de Mediana Edad , ARN Mensajero/metabolismo , Virus Vaccinia/inmunología

RESUMEN

We describe a patient with human immunodeficiency virus type-1 (HIV) infection and alveolar echinococcosis (AE) with a focus on two messages. Despite being severely immunocompromised over years the patient exhibited a long-term asymptomatic course of AE. This is in clear contrast to reports describing accelerated courses of AE in immunocompromised patients. The patient had therapeutic mebendazole drug levels with only 1/10 of the normal drug dose. He was co-treated with protease inhibitors for his HIV infection. These drugs are known as strong inhibitors of cytochrome P450 3A4 (CYP3A4)-dependent metabolism. We speculate that benzimidazoles and protease inhibitors interfere at the CYP3A4-level. The first report of co-infection of HIV and accelerated AE was in a young girl with an extremely low CD4 cell count and an abrogated lymphoproliferative responsiveness to parasite antigen stimulation. Since the CD4 cell count in our patient remained in the range of 27-150 cells/microl, we speculate that there was a critical threshold of immunosupression for constraining AE. Initial treatment with albendazole for AE added to the current highly active antiretroviral treatment (HAART), and suppressive toxoplasmosis therapy became complicated by pancytopenia. After full recovery of the bone marrow, mebendazole was introduced with a new HAART and the previously prescribed toxoplasmosis maintenance therapy. Surprisingly, efficient mebendazole levels were achieved with an uncommonly low dose. These observations suggest that the benzimidazoles, albendazole and mebendazole, may interact with protease inhibitors, which are known for their strong inhibition of the CYP3A4.

Asunto(s)

Infecciones Oportunistas Relacionadas con el SIDA/parasitología , Equinococosis Hepática/etiología , Infecciones Oportunistas Relacionadas con el SIDA/diagnóstico , Adulto , Albendazol/uso terapéutico , Antihelmínticos/uso terapéutico , Terapia Antirretroviral Altamente Activa , Equinococosis Hepática/diagnóstico , Equinococosis Hepática/tratamiento farmacológico , VIH-1 , Humanos , Masculino , Mebendazol/uso terapéutico

RESUMEN

Nitric oxide (NO) produced by the inducible form of nitric oxide synthase (iNOS) has bactericidal and virocidal effects. Although NO synthesis and iNOS expression in macrophages affect several aspects of human immunodeficiency virus (HIV) type-1 pathogenesis, their role in HIV disease remains largely unknown. In humans, the expression of iNOS is influenced by a functional CCTTT-repeat polymorphism in the promoter region of the gene. We investigated the association of this polymorphism with HIV pathogenesis in naive HIV-infected patients before the initiation of antiretroviral therapy. The allele frequencies of the iNOS CCTTT-repeat polymorphism were assessed by PCR in 857 patients from the Swiss HIV Cohort Study, including rapid progressors and long-term nonprogressors, and in 240 healthy volunteers. In HIV-infected patients, the initial viral load and the decline in total CD4 cells was calculated to estimate disease progression. Allele frequencies of the iNOS CCTTT-repeat polymorphism were similar between the HIV-infected and noninfected blood donors. In treatment-naive HIV-positive patients, there was no association of the iNOS polymorphism with viral load or with the course of CD4 cells. Regulation of iNOS expression by the functional CCTTT-polymorphism does not modify HIV pathogenesis.

Asunto(s)

Infecciones por VIH/etiología , VIH-1/patogenicidad , Óxido Nítrico Sintasa/genética , Polimorfismo Genético , Regiones Promotoras Genéticas , Adulto , Linfocitos T CD4-Positivos/inmunología , Estudios de Casos y Controles , Progresión de la Enfermedad , Frecuencia de los Genes , Infecciones por VIH/inmunología , Infecciones por VIH/metabolismo , Humanos , Modelos Lineales , Óxido Nítrico Sintasa de Tipo II , Reacción en Cadena de la Polimerasa/métodos , Carga Viral

RESUMEN

Progress in developing a small animal model of human immunodeficiency virus type 1 (HIV-1) disease would greatly facilitate studies of transmission, pathogenesis, host immune responses, and antiviral strategies. In this study, we have explored the potential of rats as a susceptible host. In a single replication cycle, rat cell lines Rat2 and Nb2 produced infectious virus at levels 10- to 60-fold lower than those produced by human cells. Rat-derived cells supported substantial levels of early HIV-1 gene expression, which was further enhanced by overexpression of human cyclin T1. Rat cells displayed quantitative, qualitative, and cell-type-specific limitations in the late phase of the HIV-1 replication cycle including relative expression levels of HIV-1 Gag proteins, intracellular Gag processing, and viral egress. Nb2 cells were rendered permissive to HIV-1 R5 viruses by coexpression of human CD4 and CCR5, indicating that the major restriction on HIV-1 replication was at the level of cellular entry. We also found that primary rat lymphocytes, macrophages, and microglia expressed considerable levels of early HIV-1 gene products following infection with pseudotyped HIV-1. Importantly, primary rat macrophages and microglia, but not lymphocytes, also expressed substantial levels of HIV-1 p24 CA and produced infectious virions. Collectively, these results identify the rat as a promising candidate for a transgenic small animal model of HIV-1 infection and highlight pertinent cell-type-specific restrictions that are features of this species.

Asunto(s)

Infecciones por VIH/virología , VIH-1/fisiología , Macrófagos/virología , Glicoproteínas de Membrana , Microglía/virología , Linfocitos T/virología , Replicación Viral , Animales , Antígenos CD4/metabolismo , Línea Celular , Células Cultivadas , Ciclina T , Ciclinas/metabolismo , Modelos Animales de Enfermedad , Duplicado del Terminal Largo de VIH/fisiología , VIH-1/genética , Humanos , Ratones , Ratas , Receptores CCR5/metabolismo , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/metabolismo , Proteínas Virales/genética , Proteínas Virales/metabolismo

RESUMEN

Human infections by Marburg (MBG) and Ebola (EBO) viruses result in lethal hemorrhagic fever. To identify cellular entry factors employed by MBG virus, noninfectible cells transduced with an expression library were challenged with a selectable pseudotype virus packaged by MBG glycoproteins (GP). A cDNA encoding the folate receptor-alpha (FR-alpha) was recovered from cells exhibiting reconstitution of viral entry. A FR-alpha cDNA was recovered in a similar strategy employing EBO pseudotypes. FR-alpha expression in Jurkat cells facilitated MBG or EBO entry, and FR-blocking reagents inhibited infection by MBG or EBO. Finally, FR-alpha bound cells expressing MBG or EBO GP and mediated syncytia formation triggered by MBG GP. Thus, FR-alpha is a significant cofactor for cellular entry for MBG and EBO viruses.

Asunto(s)

Proteínas Portadoras/fisiología , Ebolavirus/fisiología , Marburgvirus/fisiología , Receptores de Superficie Celular/fisiología , Receptores Virales/fisiología , Animales , Proteínas Portadoras/genética , Fusión Celular , Línea Celular , Chlorocebus aethiops , ADN Complementario , Receptores de Folato Anclados a GPI , Biblioteca de Genes , Células Gigantes/ultraestructura , Células Gigantes/virología , VIH-1/fisiología , Humanos , Células Jurkat , Osteosarcoma , Reacción en Cadena de la Polimerasa , Receptores de Superficie Celular/genética , Transfección , Células Tumorales Cultivadas , Células Vero , Proteínas Virales/genética

RESUMEN

OBJECTIVES: To investigate HIV trapping mechanisms in patients with acute infection and in asymptomatic individuals prior to and during antiretroviral therapy. To determine the role of complement receptor (CR), Fc gamma receptor II (Fc gammaRII), tumour necrosis factor alpha (TNFalpha), and lymphotoxin alpha (LTalpha) expression in HIV trapping efficiency. METHODS: Lymphoid tissues from three acutely HIV-infected patients and six asymptomatic, chronically HIV-infected patients collected prior to and during antiretroviral therapy were compared with lymphoid tissues from six HIV-seronegative subjects. HIV, TNFalpha and LTalpha RNA expression was detected and quantified by fluorescence in situ hybridization. CR, Fc gammaRII and HIV p24 antigen were detected and quantified by fluorescence immunohistochemistry. RESULTS: The amount of trapped HIV did not differ significantly between patients with acute HIV infection and asymptomatic individuals, and was independent of the presence of CR or Fc gammaRII expression. However, in patients with acute infection, the amount of trapped virus was correlated inversely with the number of HIV-infected cells (P = 0.0092) and with the size of the light zone (P = 0.037). In these patients, the number of TNFalpha-expressing cells was correlated inversely with the amount of trapped virus (P = 0.014) and positively correlated with the size of the light zone in germinal centers (P = 0.041). No correlations were observed between TNFalpha or LTalpha expression and Fc gammaRII or CR expression. CONCLUSION: This report provides the first evidence that in humans TNFalpha is involved in the development of lymphoid follicles, HIV trapping, and, consequently, in early host immune responses. A model is proposed for early events in patients during acute HIV infection.

Asunto(s)

Infecciones por VIH/inmunología , Infecciones por VIH/virología , VIH-1/inmunología , Tejido Linfoide/virología , Linfotoxina-alfa/fisiología , Receptores de Complemento/metabolismo , Receptores de IgG/metabolismo , Factor de Necrosis Tumoral alfa/fisiología , Enfermedad Aguda , Fármacos Anti-VIH/farmacología , Fármacos Anti-VIH/uso terapéutico , Terapia Antirretroviral Altamente Activa , Western Blotting , Enfermedad Crónica , Centro Germinal/efectos de los fármacos , Centro Germinal/inmunología , Centro Germinal/metabolismo , Centro Germinal/virología , Anticuerpos Anti-VIH/biosíntesis , Anticuerpos Anti-VIH/inmunología , Infecciones por VIH/tratamiento farmacológico , Seropositividad para VIH/tratamiento farmacológico , Seropositividad para VIH/inmunología , Seropositividad para VIH/virología , VIH-1/efectos de los fármacos , VIH-1/genética , VIH-1/fisiología , Humanos , Inmunohistoquímica , Hibridación in Situ , Tejido Linfoide/efectos de los fármacos , Tejido Linfoide/inmunología , Tejido Linfoide/metabolismo , Linfotoxina-alfa/genética , Modelos Inmunológicos , ARN Viral/análisis , ARN Viral/genética , Receptores de IgG/biosíntesis , Factor de Necrosis Tumoral alfa/genética , Carga Viral

RESUMEN

The surface molecule CD4 plays a key role in initiating cellular entry by the human immunodeficiency virus type 1 (HIV-1), and it is now recognized as acting synergistically with select chemokine receptors (coreceptors) in the infection process. The present study was undertaken to determine whether the extracellular region of CD4 is sufficient to induce fusion of HIV-1 virions with target cells in the absence of its anchoring function. Using pseudotype reporter viruses to quantitate infection, soluble CD4 (sCD4) was tested for its ability to induce fusion by viruses utilizing CCR5 as their coreceptor. We found that sCD4 was competent to replace membrane-bound CD4 to trigger infection mediated by several HIV-1 envelopes. Furthermore, in a comparison of the envelopes of HIV-1 NL4-3 and a chimera containing the gp120 V3 loop of Ba-L, the V3 region was found to be one factor affecting susceptibility to induction by sCD4. In addition, using truncated and mutant derivatives of sCD4, the amino-terminal D1 domain of CD4 was found to be necessary and sufficient for induction of fusion and to require an intact gp120-binding site for this activity. These results delineate determinants on CD4 and gp120 required for fusion induction in collaboration with a coreceptor, and suggest a mechanism whereby CD4 may contribute to viral infection in trans.

Asunto(s)

Antígenos CD4/química , Antígenos CD4/metabolismo , VIH-1/patogenicidad , Receptores CCR5/metabolismo , Antígenos CD4/genética , Línea Celular , Proteína gp120 de Envoltorio del VIH/metabolismo , VIH-1/fisiología , Células HeLa , Humanos , Fusión de Membrana , Fragmentos de Péptidos/metabolismo , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Replicación Viral

RESUMEN

The present study sought to determine how usage of coreceptors by human immunodeficiency virus type 1 dictates cell tropism and depletion of CD4(+) T cells in human lymphoid tissues cultured ex vivo. We found that coreceptor preferences control the marked, preferential depletion of coreceptor-expressing CD4(+) lymphocytes. In addition, there was a strong, but not absolute, preference shown by CXCR4-using strains for lymphocytes and by CCR5-using strains for macrophages.

Asunto(s)

Linfocitos T CD4-Positivos/metabolismo , VIH-1/metabolismo , Depleción Linfocítica , Receptores CCR5/metabolismo , Receptores CXCR4/metabolismo , Linfocitos T CD4-Positivos/virología , VIH-1/fisiología , Humanos , Tejido Linfoide/citología

RESUMEN

Since the Marburg (MBG) and Ebola (EBO) viruses have sequence homology and cause similar diseases, we hypothesized that they associate with target cells by similar mechanisms. Pseudotype viruses prepared with a luciferase-containing human immunodeficiency virus type 1 backbone and packaged by the MBG virus or the Zaire subtype EBO virus glycoproteins (GP) mediated infection of a comparable wide range of mammalian cell types, and both were inhibited by ammonium chloride. In contrast, they exhibited differential sensitivities to treatment of target cells with tunicamycin, endoglycosidase H, or protease (pronase). Therefore, while they exhibit certain functional similarities, the MBG and EBO virus GP interact with target cells by distinct processes.

Asunto(s)

Ebolavirus/patogenicidad , Marburgvirus/patogenicidad , Proteínas del Envoltorio Viral/metabolismo , Cloruro de Amonio/farmacología , Animales , Línea Celular , Ebolavirus/fisiología , VIH-1/enzimología , VIH-1/genética , Fiebre Hemorrágica Ebola/virología , Humanos , Luciferasas/genética , Enfermedad del Virus de Marburg/virología , Marburgvirus/fisiología , Proteínas del Envoltorio Viral/genética , Virulencia

RESUMEN

The chemokine receptors CCR5 and CXCR4 function as the principal coreceptors for human immunodeficiency virus type 1 (HIV-1). Coreceptor function has also been demonstrated for a variety of related receptors in vitro. The relative contributions of CCR5, CXCR4, and other putative coreceptors to HIV-1 disease in vivo have yet to be defined. In this study, we used sequential primary isolates and recombinant strains of HIV-1 to demonstrate that CXCR4-using (X4) viruses emerging in association with disease progression are highly pathogenic in ex vivo lymphoid tissues compared to CXCR4-independent viruses. Furthermore, synthetic receptor antagonists that specifically block CXCR4-mediated entry dramatically suppressed the depletion of CD4(+) T cells by recombinant and clinically derived X4 HIV-1 isolates. Moreover, in vitro specificity for the additional coreceptors CCR3, CCR8, BOB, and Bonzo did not augment cytopathicity or diminish sensitivity toward CXCR4 antagonists in lymphoid tissues. These data provide strong evidence to support the concept that adaptation to CXCR4 specificity in vivo accelerates HIV-1 disease progression. Thus, therapeutic intervention targeting the interaction of HIV-1 gp120 with CXCR4 may be highly valuable for suppressing the pathogenic effects of late-stage viruses.

Asunto(s)

VIH-1/fisiología , Fusión de Membrana/fisiología , Receptores CXCR4/fisiología , Animales , Linfocitos T CD4-Positivos/citología , Células COS , Línea Celular , VIH-1/patogenicidad , Humanos , Depleción Linfocítica , Virulencia , Replicación Viral

RESUMEN

Chemokine receptors, particularly CCR5 and CXCR4, act as essential coreceptors in concert with CD4 for cellular entry by human immunodeficiency virus type 1 (HIV-1; reviewed in [1]). But infection of CD4(-) cells has also been encountered in various tissues in vivo, including astrocytes, neurons and microvascular endothelial cells of the brain [2] [3] [4] [5] [6], epithelial cells [5] [7], CD4(-) lymphocytes and thymocytes [8] [9], and cardiomyocytes [10]. Here, we present evidence for the infection of CD4(-) cell lines bearing coreceptors by well-known HIV-1 strains when co-cultured with CD4(+) cells. This process requires contact between the coreceptor-bearing and CD4(+) cells and supports the full viral replication cycle within the coreceptor-bearing target cell. Furthermore, CD4 provided in trans facilitates infection of primary human cells, such as brain-derived astrocytes. Although the pathobiological significance of infection of CD4(-) cells in vivo remains to be elucidated, this trans-receptor mechanism may facilitate generation of hidden reservoirs of latent virus that confound antiviral therapies and that contribute to specific AIDS-associated clinical syndromes.

Asunto(s)

Antígenos CD4/análisis , VIH-1/fisiología , Fusión de Membrana/fisiología , Receptores del VIH/fisiología , Línea Celular , Separación Celular , Citometría de Flujo , Humanos