RESUMEN
BACKGROUND: Novel treatments are needed for Staphylococcus aureus bacteremia, particularly for methicillin-resistant S. aureus (MRSA). Exebacase is a first-in-class antistaphylococcal lysin that is rapidly bactericidal and synergizes with antibiotics. METHODS: In Direct Lysis of Staph Aureus Resistant Pathogen Trial of Exebacase (DISRUPT), a superiority-design phase 3 study, patients with S. aureus bacteremia/endocarditis were randomly assigned to receive a single dose of intravenous exebacase or placebo in addition to standard-of-care antibiotics. The primary efficacy outcome was clinical response at day 14 in the MRSA population. RESULTS: A total of 259 patients were randomized before the study was stopped for futility based on the recommendation of the unblinded Data Safety Monitoring Board. Clinical response rates at day 14 in the MRSA population (n = 97) were 50.0% (exebacase + antibiotics; 32/64) versus 60.6% (antibiotics alone; 20/33) (P = .392). Overall, rates of adverse events were similar across groups. No adverse events of hypersensitivity related to exebacase were reported. CONCLUSIONS: Exebacase + antibiotics failed to improve clinical response at day 14 in patients with MRSA bacteremia/endocarditis. This result was unexpected based on phase 2 data that established proof-of-concept for exebacase + antibiotics in patients with MRSA bacteremia/endocarditis. In the antibiotics-alone group, the clinical response rate was higher than that seen in phase 2. Heterogeneity within the study population and a relatively small sample size in either the phase 2 or phase 3 studies may have increased the probability of imbalances in the multiple components of day 14 clinical outcome. This study provides lessons for future superiority studies in S. aureus bacteremia/endocarditis. Clinical Trials Registration.NCT04160468.
Asunto(s)
Antibacterianos , Bacteriemia , Staphylococcus aureus Resistente a Meticilina , Infecciones Estafilocócicas , Humanos , Infecciones Estafilocócicas/tratamiento farmacológico , Infecciones Estafilocócicas/microbiología , Masculino , Femenino , Antibacterianos/uso terapéutico , Antibacterianos/administración & dosificación , Persona de Mediana Edad , Bacteriemia/tratamiento farmacológico , Bacteriemia/microbiología , Anciano , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Adulto , Endocarditis Bacteriana/tratamiento farmacológico , Endocarditis Bacteriana/microbiología , Resultado del Tratamiento , Nivel de Atención , Quimioterapia Combinada , Staphylococcus aureus/efectos de los fármacosRESUMEN
BACKGROUND: Recurrent Clostridioides difficile infection (rCDI) is associated with loss of microbial diversity and microbe-derived secondary bile acids, which inhibit C. difficile germination and growth. SER-109, an investigational microbiome drug of donor-derived, purified spores, reduced recurrence in a dose-ranging, phase (P) 1 study in subjects with multiple rCDIs. METHODS: In a P2 double-blind trial, subjects with clinical resolution on standard-of-care antibiotics were stratified by age (< or ≥65 years) and randomized 2:1 to single-dose SER-109 or placebo. Subjects were diagnosed at study entry by PCR or toxin testing. Safety, C. difficile-positive diarrhea through week 8, SER-109 engraftment, and bile acid changes were assessed. RESULTS: 89 subjects enrolled (67% female; 80.9% diagnosed by PCR). rCDI rates were lower in the SER-109 arm than placebo (44.1% vs 53.3%) but did not meet statistical significance. In a preplanned analysis, rates were reduced among subjects ≥65 years (45.2% vs 80%, respectively; RR, 1.77; 95% CI, 1.11-2.81), while the <65 group showed no benefit. Early engraftment of SER-109 was associated with nonrecurrence (P < .05) and increased secondary bile acid concentrations (P < .0001). Whole-metagenomic sequencing from this study and the P1 study revealed previously unappreciated dose-dependent engraftment kinetics and confirmed an association between early engraftment and nonrecurrence. Engraftment kinetics suggest that P2 dosing was suboptimal. Adverse events were generally mild to moderate in severity. CONCLUSIONS: Early SER-109 engraftment was associated with reduced CDI recurrence and favorable safety was observed. A higher dose of SER-109 and requirements for toxin testing were implemented in the current P3 trial. CLINICAL TRIALS REGISTRATION: NCT02437487, https://clinicaltrials.gov/ct2/show/NCT02437487?term=SER-109&draw= 2&rank=4.
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Clostridioides difficile , Infecciones por Clostridium , Microbiota , Anciano , Clostridioides , Infecciones por Clostridium/tratamiento farmacológico , Infecciones por Clostridium/prevención & control , Drogas en Investigación , Femenino , Humanos , Masculino , RecurrenciaRESUMEN
BACKGROUND & AIMS: Firmicutes bacteria produce metabolites that maintain the intestinal barrier and mucosal immunity. Firmicutes are reduced in the intestinal microbiota of patients with ulcerative colitis (UC). In a phase 1b trial of patients with UC, we evaluated the safety and efficacy of SER-287, an oral formulation of Firmicutes spores, and the effects of vancomycin preconditioning on expansion (engraftment) of SER-287 species in the colon. METHODS: We conducted a double-blind trial of SER-287 in 58 adults with active mild-to-moderate UC (modified Mayo scores 4-10, endoscopic subscores ≥1). Participants received 6 days of preconditioning with oral vancomycin (125 mg, 4 times daily) or placebo followed by 8 weeks of oral SER-287 or placebo. Patients were randomly assigned (2:3:3:3) to groups that received placebo followed by either placebo or SER-287 once weekly, or vancomycin followed by SER-287 once weekly, or SER-287 once daily. Clinical end points included safety and clinical remission (modified Mayo score ≤2; endoscopic subscores 0 or 1). Microbiome end points included SER-287 engraftment (dose species detected in stool after but not before SER-287 administration). Engraftment of SER-287 and changes in microbiome composition and associated metabolites were measured by analyses of stool specimens collected at baseline, after preconditioning, and during and 4 weeks after administration of SER-287 or placebo. RESULTS: Proportions of patients with adverse events did not differ significantly among groups. A higher proportion of patients in the vancomycin/SER-287 daily group (40%) achieved clinical remission at week 8 than patients in the placebo/placebo group (0%), placebo/SER-287 weekly group (13.3%), or vancomycin/SER-287 weekly group (17.7%) (P = .024 for vancomycin/SER-287 daily vs placebo/placebo). By day 7, higher numbers of SER-287 dose species were detected in stool samples from all SER-287 groups compared with the placebo group (P < .05), but this difference was not maintained beyond day 7 in the placebo/SER-287 weekly group. In the vancomycin groups, a greater number of dose species were detected in stool collected on day 10 and all subsequent time points through 4 weeks post dosing compared with the placebo group (P < .05). A higher number of SER-287 dose species were detected in stool samples on days 7 and 10 from subjects who received daily vs weekly SER-287 doses (P < .05). Changes in fecal microbiome composition and metabolites were associated with both vancomycin/SER-287 groups. CONCLUSIONS: In this small phase 1b trial of limited duration, the safety and tolerability of SER-287 were similar to placebo. SER-287 after vancomycin was significantly more effective than placebo for induction of remission in patients with active mild to moderate UC. Engraftment of dose species was facilitated by vancomycin preconditioning and daily dosing of SER-287. ClinicalTrials.gov ID NCT02618187.
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Colitis Ulcerosa/terapia , Firmicutes , Microbioma Gastrointestinal , Adulto , Método Doble Ciego , Femenino , Humanos , Masculino , Persona de Mediana Edad , EsporasRESUMEN
BACKGROUNDNovel therapeutic approaches are critically needed for Staphylococcus aureus bloodstream infections (BSIs), particularly for methicillin-resistant S. aureus (MRSA). Exebacase, a first-in-class antistaphylococcal lysin, is a direct lytic agent that is rapidly bacteriolytic, eradicates biofilms, and synergizes with antibiotics.METHODSIn this superiority-design study, we randomly assigned 121 patients with S. aureus BSI/endocarditis to receive a single dose of exebacase or placebo. All patients received standard-of-care antibiotics. The primary efficacy endpoint was clinical outcome (responder rate) on day 14.RESULTSClinical responder rates on day 14 were 70.4% and 60.0% in the exebacase + antibiotics and antibiotics-alone groups, respectively (difference = 10.4, 90% CI [-6.3, 27.2], P = 0.31), and were 42.8 percentage points higher in the prespecified exploratory MRSA subgroup (74.1% vs. 31.3%, difference = 42.8, 90% CI [14.3, 71.4], ad hoc P = 0.01). Rates of adverse events (AEs) were similar in both groups. No AEs of hypersensitivity to exebacase were reported. Thirty-day all-cause mortality rates were 9.7% and 12.8% in the exebacase + antibiotics and antibiotics-alone groups, respectively, with a notable difference in MRSA patients (3.7% vs. 25.0%, difference = -21.3, 90% CI [-45.1, 2.5], ad hoc P = 0.06). Among MRSA patients in the United States, median length of stay was 4 days shorter and 30-day hospital readmission rates were 48% lower in the exebacase-treated group compared with antibiotics alone.CONCLUSIONThis study establishes proof of concept for exebacase and direct lytic agents as potential therapeutics and supports conduct of a confirmatory study focused on exebacase to treat MRSA BSIs.TRIAL REGISTRATIONClinicaltrials.gov NCT03163446.FUNDINGContraFect Corporation.
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Endocarditis Bacteriana , Endopeptidasas/administración & dosificación , Staphylococcus aureus Resistente a Meticilina/metabolismo , Infecciones Estafilocócicas , Adulto , Supervivencia sin Enfermedad , Endocarditis Bacteriana/sangre , Endocarditis Bacteriana/tratamiento farmacológico , Endocarditis Bacteriana/mortalidad , Femenino , Humanos , Masculino , Infecciones Estafilocócicas/sangre , Infecciones Estafilocócicas/tratamiento farmacológico , Infecciones Estafilocócicas/mortalidad , Tasa de SupervivenciaRESUMEN
BACKGROUND: Patients with recurrent Clostridium difficile infection (CDI) have a ≥60% risk of relapse, as conventional therapies do not address the underlying gastrointestinal dysbiosis. This exploratory study evaluated the safety and efficacy of bacterial spores for preventing recurrent CDI. METHODS: Stool specimens from healthy donors were treated with ethanol to eliminate pathogens. The resulting spores were fractionated and encapsulated for oral delivery as SER-109. Following their response to standard-of-care antibiotics, patients in cohort 1 were treated with SER-109 on 2 consecutive days (geometric mean dose, 1.7 × 10(9) spores), and those in cohort 2 were treated on 1 day (geometric mean dose, 1.1 × 10(8) spores). The primary efficacy end point was absence of C. difficile-positive diarrhea during an 8-week follow-up period. Microbiome alterations were assessed. RESULTS: Thirty patients (median age, 66.5 years; 67% female) were enrolled, and 26 (86.7%) met the primary efficacy end point. Three patients with early, self-limiting C. difficile-positive diarrhea did not require antibiotics and tested negative for C. difficile at 8 weeks; thus, 96.7% (29 of 30) achieved clinical resolution. In parallel, gut microbiota rapidly diversified, with durable engraftment of spores and no outgrowth of non-spore-forming bacteria found after SER-109 treatment. Adverse events included mild diarrhea, abdominal pain, and nausea. CONCLUSIONS: SER-109 successfully prevented CDI and had a favorable safety profile, supporting a novel microbiome-based intervention as a potential therapy for recurrent CDI.
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Terapia Biológica/métodos , Clostridioides difficile/crecimiento & desarrollo , Infecciones por Clostridium/prevención & control , Microbioma Gastrointestinal , Tracto Gastrointestinal/microbiología , Prevención Secundaria/métodos , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Terapia Biológica/efectos adversos , Diarrea/prevención & control , Femenino , Humanos , Masculino , Persona de Mediana Edad , Adulto JovenRESUMEN
Genetically engineered retroviruses are widely used for gene delivery into human cells. A number of investigators have studied spleen necrosis virus (SNV) as a vehicle for gene delivery. Vectors developed from SNV and its closely associated avian reticuloendotheliosis virus strain A (REV-A) can be used for gene transfer into a variety of cells, including primary hematopoietic cells and human brain and post-mitotic neuronal cells that are difficult to transduce with other vector systems. SNV-based vector systems have the advantage of being quite safe, because wild-type SNV is unable to infect human cells and has less preference for integration into transcriptionally active sites or genes. However, the generation of retroviral vectors requires cotransfection of more than one plasmid into a packaging cell line, which is a tedious process. The development of stable packaging cell lines expressing envelope (Env) proteins and the structural proteins Gag-Pol will enhance mass production of retroviral vectors for future gene therapy experiments both in vitro and in vivo. This protocol describes the generation of retroviral particles for the SNV-based vector system. These particles can then be used for transduction of various cell types; as an example, a technique for transduction of post-mitotic neurons is also presented.
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Vectores Genéticos/genética , Virus de la Necrosis Esplénica del Pato de Trager/genética , Transducción Genética/métodos , Virión/genética , Línea Celular , Humanos , Mitosis , Neuronas/citología , Neuronas/metabolismo , Fracciones Subcelulares/metabolismo , TransfecciónRESUMEN
Hyperglycemic conditions associated with diabetes mellitus (DM) or with the use of antiretroviral therapy may increase the risk of central nervous system (CNS) disorders in HIV-1 infected patients. In support of this hypothesis, we investigated the combined effects of hyperglycemic conditions and HIV-1 accessory protein Nef on the CNS using both in vitro and in vivo models. Astrocytes, the most abundant glial cell type required for normal synaptic transmission and other functions were selected for our in vitro study. The results show that in vitro hyperglycemic conditions enhance the expression of proinflammatory cytokines including caspase-3, complement factor 3 (C3), and the production of total nitrate and 8-iso-PGF2 alpha as reactive oxygen species (ROS) in human astrocytes leading to cell death in a dose-dependent manner. Delivery of purified recombinant HIV-1 Nef protein, or Nef expressed via HIV-1-based vectors in astrocytes showed similar results. The expression of Nef protein delivered via HIV-1 vectors in combination with hyperglycemia further augmented the production of ROS, C3, activation of caspase-3, modulation of filamentous protein (F-protein), depolarization of the mitochondria, and loss of astrocytes. To further verify the effects of hyperglycemia and HIV-1 Nef protein on CNS individually or in combination, in vivo studies were performed in streptozotocin (STZ) induced diabetic mice, by injecting HIV-1 Nef expressing viral particles into the sub-cortical region of the brain. Our in vivo results were similar to in vitro findings indicating an enhanced production of caspases-3, ROS (lipid oxidation and total nitrate), and C3 in the brain tissues of these animals. Interestingly, the delivery of HIV-1 Nef protein alone caused similar damage to CNS as augmented by hyperglycemia conditions. Taken together, the data suggests that HIV-1 infected individuals with hyperglycemia could potentially be at a higher risk of developing CNS related complications.
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Complicaciones de la Diabetes , Infecciones por VIH/complicaciones , VIH-1/patogenicidad , Factores de Virulencia/fisiología , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/fisiología , Animales , Apoptosis , Astrocitos/patología , Infecciones por VIH/patología , Mediadores de Inflamación/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NODRESUMEN
Although combination therapy for HIV infection represents a triumph for modern medicine, chronic suppressive therapy is required to contain persistent infection in reservoirs such as latently infected CD4+ lymphocytes and cells of the macrophage-monocyte lineage. Despite its success, chronic suppressive therapy is limited by its cost, the requirement of lifelong adherence, and the unknown effects of long-term treatment. This review discusses our current understanding of suppressive antiretroviral therapy, the latent viral reservoir, and the needs for and challenges of attacking this reservoir to achieve a cure.
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Fármacos Anti-VIH/uso terapéutico , Infecciones por VIH/tratamiento farmacológico , VIH/efectos de los fármacos , Latencia del Virus/efectos de los fármacos , Vacunas contra el SIDA , Animales , Fármacos Anti-VIH/economía , Terapia Antirretroviral Altamente Activa/efectos adversos , Terapia Antirretroviral Altamente Activa/economía , Linfocitos T CD4-Positivos/virología , Ensayos Clínicos como Asunto , Descubrimiento de Drogas , VIH/fisiología , Infecciones por VIH/virología , Humanos , Viremia/tratamiento farmacológico , Replicación Viral/efectos de los fármacosRESUMEN
The catabolic pathway of l-tryptophan (l-trp), known as the kynurenine pathway (KP), has been implicated in the pathogenesis of a wide range of brain diseases through its ability to lead to immune tolerance and neurotoxicity. As endothelial cells (ECs) and pericytes of the blood-brain-barrier (BBB) are among the first brain-associated cells that a blood-borne pathogen would encounter, we sought to determine their expression of the KP. Using RT-PCR and HPLC/GC-MS, we show that BBB ECs and pericytes constitutively express components of the KP. BBB ECs constitutively synthesized kynurenic acid, and after immune activation, kynurenine (KYN), which is secreted basolaterally. BBB pericytes produced small amounts of picolinic acid and after immune activation, KYN. These results have significant implications for the pathogenesis of inflammatory brain diseases in general, particularly human immunodeficiency virus (HIV)-related brain disease. Kynurenine pathway activation at the BBB results in local immune tolerance and neurotoxicity: the basolateral secretion of excess KYN can be further metabolized by perivascular macrophages and microglia with synthesis of quinolinic acid. The results point to a mechanism whereby a systemic inflammatory signal can be transduced across an intact BBB to cause local neurotoxicity.
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Barrera Hematoencefálica/metabolismo , Tolerancia Inmunológica , Quinurenina/fisiología , Síndromes de Neurotoxicidad/metabolismo , Transducción de Señal/inmunología , Barrera Hematoencefálica/inmunología , Barrera Hematoencefálica/patología , Células Cultivadas , Células Endoteliales/inmunología , Células Endoteliales/metabolismo , Células Endoteliales/patología , Humanos , Quinurenina/genética , Quinurenina/metabolismo , Síndromes de Neurotoxicidad/genética , Síndromes de Neurotoxicidad/patología , Pericitos/inmunología , Pericitos/metabolismo , Pericitos/patologíaRESUMEN
The success of highly active antiretroviral therapy (HAART) for HIV-1 infection has sparked interest in mechanisms by which the virus can persist despite effectively suppressive therapy. Latent HIV-1 reservoirs established early during infection not only prevent sterilizing immunity but also represent a major obstacle to virus eradication. When HIV-1 gains a foothold in the immunologic memory or in certain inaccessible compartments of the human body, it cannot be easily purged by HAART and is able to replenish systemic infection on treatment interruption. Because latently infected cells are indistinguishable from uninfected cells, deliberate activation of latent infection combined with intensified HAART seems to be the best strategy to combat latent infection. Initial hypothesis-driven clinical trials did not achieve their ultimate goal, although they provided valuable insight for the design of future eradication protocols. A more detailed understanding of the basic mechanisms underlying the establishment and long-term maintenance of HIV-1 reservoirs will be critical in developing new eradication approaches.
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Terapia Antirretroviral Altamente Activa , Infecciones por VIH/tratamiento farmacológico , VIH-1/fisiología , Humanos , Insuficiencia del Tratamiento , Activación Viral , Latencia del VirusRESUMEN
Medicinal plants have been widely used to treat a variety of infectious and non-infectious ailments. According to one estimate, 25% of the commonly used medicines contain compounds isolated from plants. Several plants could offer a rich reserve for drug discovery of infectious diseases, particularly in an era when the latest separation techniques are available on one hand, and the human population is challenged by a number of emerging infectious diseases on the other hand. Among several other ailments, viral infections, particularly infections associated with human immunodeficiency virus type 1 (HIV-1) and 2 (HIV-2), and newly emerging infectious viruses have challenged mankind survival. Of importance, a variety of medicinal plants have shown promise to treat a number of viral infections, and some of them possess broad-spectrum antiviral activity. In the past, exploration into the antiviral activity of various promising medicinal plants was limited due to: (a) highly infectious nature of viruses and (b) lack of appropriate separation techniques for the identification of antiviral components from plants. Development of vector-based strategies, in which non-infectious molecular clone of a virus could be used for antiviral screening purposes, and advancement in separation technologies offers promise for medicinal plants usage in modern drug discovery. This article describes potential antiviral properties of medicinal plants against a diverse group of viruses, and suggests screening the potential of plants possessing broad-spectrum antiviral effects against emerging viral infections.
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Antivirales/farmacología , Extractos Vegetales/farmacología , Plantas Medicinales/química , Virus/efectos de los fármacos , Antivirales/aislamiento & purificación , HumanosRESUMEN
Pentoxifylline, a caffeine-related compound, was shown to suppress human immunodeficiency virus type 1 (HIV-1) replication. This effect is thought to be mediated by inhibition of tumor necrosis factor-alpha (TNFalpha)-mediated long-terminal repeat (LTR)-driven expression. We now demonstrate that pentoxifylline efficiently inhibits transduction by HIV-1-based vectors. This latter effect is independent of LTR-driven expression, and correlates with a reduced efficiency of the completion of the integration process in infected cells. Finally, the effect of pentoxifylline is dramatically reduced in cells expressing a dominant negative ATR protein, and in primary human cells that exhibit low level of ATR activity, suggesting that the effect of pentoxifylline on HIV-1 transduction and replication is at least partly mediated by suppression of the ATR kinase.
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Antivirales/farmacología , Vectores Genéticos/efectos de los fármacos , VIH-1/efectos de los fármacos , Pentoxifilina/farmacología , Transducción Genética , Proteínas de la Ataxia Telangiectasia Mutada , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/genética , Línea Celular , Humanos , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , Integración Viral/efectos de los fármacos , Replicación Viral/efectos de los fármacosRESUMEN
The oral cavity of human immunodeficiency virus type I (HIV-1) infected individuals is subjected to a series of opportunistic infections which are usually considered as a prognostic marker for the severity of infection as well as an indicator of immunodeficiency. The highly active antiretroviral therapy (HAART) has significantly lessened the severity of HIV-associated oral infections although this therapeutic regimen is considered to be responsible for some of the oral lesions such as oral warts and salivary gland disorders. In addition, the beneficial effects of HAART on HIV associated oral lesions are stratified with age, with the adult population showing improvements whereas the oral lesions among children remain unchanged with this therapy. The presence of HIV-1 in the saliva, and infectivity of oral epithelial cells suggest that the oral cavity is a site of HIV pathogenesis and potential reservoir for the disease in the setting of virally suppressive HAART. Overall HIV associated oral lesions are usually due to fungal, bacterial, and viral infections as well as some of unknown etiology. This review describes the current status of HIV associated oral lesions by comparing historically available pre-HAART data. Future directions envisioned by the National Institutes of Health as well as novel avenues to be explored are also presented.
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Fármacos Anti-VIH/uso terapéutico , Infecciones por VIH/tratamiento farmacológico , VIH-1 , Enfermedades de la Boca/etiología , Adulto , Terapia Antirretroviral Altamente Activa , Niño , Infecciones por VIH/complicaciones , Infecciones por VIH/transmisión , HumanosRESUMEN
We analyzed the safety and immunogenicity of attenuated rabies virus vectors expressing simian-human immunodeficiency virus (SHIV)-1(89.6P) Env or simian immunodeficiency virus (SIV)(mac239) Gag in rhesus macaques. Four test macaques were immunized with both vaccine constructs, and 2 control macaques received an empty rabies vector. Seroconversion against rabies virus glycoprotein (G) and SHIV(89.6P) Env was detected after the initial immunization, but no cellular responses against SHIV antigens were observed. HIV/SIV-specific immune responses were not enhanced by boosts with the same vectors. Therefore, we constructed vectors expressing SHIV(89.6P) Env and SIV(mac239) Gag in which the rabies G was replaced with the G protein of vesicular stomatitis virus (VSV). Two years after initial immunization, a boost with the rabies-VSV G vectors resulted in SIV/HIV-specific immune responses. Upon challenge with SHIV(89.6P) test macaques controlled the infection, whereas control macaques had high levels of viremia and a profound loss of CD4(+) T cells, with 1 control macaque dying of an AIDS-like disease.
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Productos del Gen env/inmunología , Productos del Gen gag/inmunología , Vacunas contra el SIDAS/uso terapéutico , Síndrome de Inmunodeficiencia Adquirida del Simio/prevención & control , Vacunas Sintéticas/uso terapéutico , Animales , Cartilla de ADN , Productos del Gen env/genética , Productos del Gen gag/genética , Vectores Genéticos , Macaca mulatta , Masculino , ARN Viral/análisis , Virus de la Rabia/genética , Virus de la Inmunodeficiencia de los Simios/genéticaRESUMEN
The human immunodeficiency virus type I (HIV-1) accessory protein Vpr has been associated with the induction of programmed cell death (apoptosis) and cell-cycle arrest. Studies have shown the apoptotic effect of Vpr on primary and established cell lines and on diverse tissues including the central nervous system (CNS) in vitro. However, the relevance of the effect of Vpr observed in vitro to HIV-1 neuropathogenesis in vivo, remains unknown. Due to the narrow host range of HIV-1 infection, no animal model is currently available. This has prompted us to consider a small animal model to evaluate the effects of Vpr on CNS in vivo through surrogate viruses expressing HIV-1Vpr. A single round of replication competent viral vectors, expressing Vpr, were used to investigate the apoptosis-inducing capabilities of HIV-1Vpr in vivo. Viral particles pseudotyped with VSV-G or N2c envelopes were generated from spleen necrosis virus (SNV) and HIV-1-based vectors to transduce CNS cells. The in vitro studies have demonstrated that Vpr generated by SNV vectors had less apoptotic effects on CNS cells compared with Vpr expressed by HIV-1 vectors. The in vivo study has suggested that viral particles, expressing Vpr generated by HIV-1-based vectors, when delivered through the ventricle, caused loss of neurons and dendritic processes in the cortical region. The apoptotic effect was extended beyond the cortical region and affected the hippocampus neurons, the lining of the choroids plexus, and the cerebellum. However, the effect of Vpr, when delivered through the cortex, showed neuronal damage only around the site of injection. Interestingly, the number of apoptotic neurons were significantly higher with HIV-1 vectors expressing Vpr than by the SNV vectors. This may be due to the differences in the proteins expressed by these viral vectors. These results suggest that Vpr induces apoptosis in CNS cells in vitro and in vivo. To our knowledge, this is the first study to investigate the apoptosis-inducing capabilities of HIV-1Vpr in vivo in neonatal mice. We propose that this, in expensive animal model, may be of value to design-targeted neuroprotective therapeutics.
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Apoptosis , Encéfalo/patología , Productos del Gen vpr/fisiología , VIH-1/metabolismo , Animales , Animales Recién Nacidos , Encéfalo/metabolismo , Células Cultivadas , Vectores Genéticos , Humanos , Ratones , Ratones Endogámicos C57BL , Oligodendroglía/patología , Virus de la Necrosis Esplénica del Pato de Trager/genética , Productos del Gen vpr del Virus de la Inmunodeficiencia HumanaRESUMEN
The majority of human immunodeficiency virus type 1 (HIV-1)-infected individuals are either alcoholics or prone to alcoholism. Upon ingestion, alcohol is easily distributed into the various compartments of the body, particularly the brain, by crossing through the blood-brain barrier. Both HIV-1 and alcohol induce oxidative stress, which is considered a precursor for cytotoxic responses. Several reports have suggested that statins exert antioxidant as well as anti-inflammatory pleiotropic effects, besides their inherent cholesterol-depleting potentials. In our studies, postmitotically differentiated neurons were cocultured with HIV-1-infected monocytes, T cells, or their cellular supernatants in the presence of physiological concentrations of alcohol for 72 h. Parallel cultures were pretreated with statins (atorvastatin and simvastatin) with the appropriate controls, i.e., postmitotically differentiated neurons cocultured with uninfected cells and similar cultures treated with alcohol. The oxidative stress responses in the presence/absence of alcohol in these cultures were determined by the production of the well-characterized oxidative stress markers, 8-isoprostane-F2-alpha, total nitrates as an indicator for various isoforms of nitric oxide synthase activity, and heat shock protein 70 (Hsp70). An in vitro culture of postmitotically differentiated neurons with HIV-1-infected monocytes or T cells as well as supernatants from these cells enhanced the release of 8-isoprostane-F2-alpha in the conditioned medium six- to sevenfold (monocytes) and four- to fivefold (T cells). It was also observed that coculturing of HIV-1-infected primary monocytes over a time period of 72 h significantly elevated the release of Hsp70 compared with that of uninfected controls. Cellular supernatants of HIV-1-infected monocytes or T cells slightly increased Hsp70 levels compared to neurons cultured with uninfected monocytes or T-cell supernatants (controls). Ethanol (EtOH) presence further elevated Hsp70 in both infected and uninfected cultures. The amount of total nitrates was significantly elevated in the coculture system when both infected cells and EtOH were present. Surprisingly, pretreatment of postmitotic neurons with clinically available inhibitors of HMG-coenzyme A reductase (statins) inhibited HIV-1-induced release of stress/toxicity-associated parameters, i.e., Hsp70, isoprostanes, and total nitrates from HIV-1-infected cells. The results of this study provide new insights into HIV-1 neuropathogenesis aimed at the development of future HIV-1 therapeutics to eradicate viral reservoirs from the brain.
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Colesterol/metabolismo , Etanol/farmacología , VIH-1/efectos de los fármacos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Neuronas/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Depresores del Sistema Nervioso Central/farmacología , VIH-1/metabolismo , Humanos , Neuronas/metabolismoRESUMEN
An internal RNA loop, located within the packaging signal of human immunodeficiency virus 1, that resembles the Rev-responsive element (RRE) closely was identified previously. Subsequent in vitro studies confirmed that the loop, termed loop A, could bind Rev protein specifically. Its proximity to the major splice donor has suggested a role for Rev-loop A interaction supplementary to or preceding that of the Rev-RRE interaction. To investigate this further in a replication-competent provirus, loop A was mutated to decrease its affinity for Rev. Impairing the Rev-loop A interaction led to reduced nuclear export of viral genomic RNA. RNA packaging decreased by approximately 30%. Viral protein production and export of virus particles appeared normal; however, the virus was severely replication-deficient. The loop A sequence, which is 98% conserved amongst viral isolates, is implicated in several cis-acting functions critical to virus viability.
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Genes rev/genética , VIH-1/genética , Mutación/genética , Transporte de ARN , ARN Viral/metabolismo , Ensamble de Virus/fisiología , Replicación Viral/fisiología , Animales , Células COS , Chlorocebus aethiops , Regulación Viral de la Expresión Génica , Unión Proteica , ARN Viral/genética , Ensamble de Virus/genética , Replicación Viral/genéticaRESUMEN
Live viral vectors expressing foreign antigens have shown great promise as vaccines against viral diseases. However, safety concerns remain a major problem regarding the use of even highly attenuated viral vectors. Using the rabies virus (RV) envelope protein as a carrier molecule, we show here that inactivated RV particles can be utilized to present Bacillus anthracis protective antigen (PA) domain-4 in the viral membrane. In addition to the RV glycoprotein (G) transmembrane and cytoplasmic domains, a portion of the RV G ectodomain was required to express the chimeric RV G anthrax PA on the cell surface. The novel antigen was also efficiently incorporated into RV virions. Mice immunized with the inactivated recombinant RV virions exhibited seroconversion against both RV G and anthrax PA, and a second inoculation greatly increased these responses. These data demonstrate that a viral envelope protein can carry a bacterial protein and that a viral carrier can display whole polypeptides compared to the limited epitope presentation of previous viral systems.
Asunto(s)
Vacunas contra el Carbunco/administración & dosificación , Carbunco/prevención & control , Antígenos Bacterianos/genética , Antígenos Virales/genética , Bacillus anthracis/inmunología , Toxinas Bacterianas/genética , Vectores Genéticos/genética , Glicoproteínas/genética , Vacunas Antirrábicas/genética , Vacunación , Proteínas del Envoltorio Viral/genética , Animales , Carbunco/sangre , Carbunco/inmunología , Anticuerpos Antibacterianos/sangre , Anticuerpos Antivirales/sangre , Especificidad de Anticuerpos , Antígenos Bacterianos/inmunología , Antígenos Virales/inmunología , Toxinas Bacterianas/inmunología , Bioterrorismo/prevención & control , Vectores Genéticos/inmunología , Glicoproteínas/inmunología , Esquemas de Inmunización , Inyecciones Intramusculares , Recuento de Linfocitos , Linfocitos/citología , Linfocitos/inmunología , Ratones , Vacunas Antirrábicas/inmunología , Recombinación Genética , Células Th2/inmunología , Vacunas Sintéticas/administración & dosificación , Proteínas del Envoltorio Viral/inmunologíaRESUMEN
In the current study, we extended our previous works on natural endogenous reverse transcription (NERT) and further examined its potential as a virucide molecular target in sexual transmission of primate lentiviruses. HIV-1 and SIV virions were pretreated with select nucleoside (NRTIs) and nonnucleoside RT inhibitors (NNRTIs), either alone or in combination with NERT-stimulating substances. The effects of these antiretrovirals on virion inactivation were analyzed in human T cell lines and primary cell cultures. Pretreatment of HIV-1 virions with physiologic NERT-stimulants and 3'-azido-3'-deoxythymidine 5'-triphosphate (AZT-TP) or nevirapine potently inactivated cell-free HIV-1 virions and resulted in strong inhibition of the viral infectivity. Pretreatment of chimeric SHIV-RT virions with NERT-stimulating cocktail and select antiretrovirals also resulted in virion inactivation and inhibition of viral infectivity in T cell lines. Our findings demonstrate the potential clinical utility of approaches based on inhibiting NERT in sexual transmission of HIV-1, through the development of effective anti-HIV-1 microbicides, such as NRTIs and NNRTIs.