RESUMEN
BACKGROUND: We previously showed that small interfering RNAs (siRNAs) targeting the Zaire Ebola virus (ZEBOV) RNA polymerase L protein formulated in stable nucleic acid-lipid particles (SNALPs) completely protected guineapigs when administered shortly after a lethal ZEBOV challenge. Although rodent models of ZEBOV infection are useful for screening prospective countermeasures, they are frequently not useful for prediction of efficacy in the more stringent non-human primate models. We therefore assessed the efficacy of modified non-immunostimulatory siRNAs in a uniformly lethal non-human primate model of ZEBOV haemorrhagic fever. METHODS: A combination of modified siRNAs targeting the ZEBOV L polymerase (EK-1 mod), viral protein (VP) 24 (VP24-1160 mod), and VP35 (VP35-855 mod) were formulated in SNALPs. A group of macaques (n=3) was given these pooled anti-ZEBOV siRNAs (2 mg/kg per dose, bolus intravenous infusion) after 30 min, and on days 1, 3, and 5 after challenge with ZEBOV. A second group of macaques (n=4) was given the pooled anti-ZEBOV siRNAs after 30 min, and on days 1, 2, 3, 4, 5, and 6 after challenge with ZEBOV. FINDINGS: Two (66%) of three rhesus monkeys given four postexposure treatments of the pooled anti-ZEBOV siRNAs were protected from lethal ZEBOV infection, whereas all macaques given seven postexposure treatments were protected. The treatment regimen in the second study was well tolerated with minor changes in liver enzymes that might have been related to viral infection. INTERPRETATION: This complete postexposure protection against ZEBOV in non-human primates provides a model for the treatment of ZEBOV-induced haemorrhagic fever. These data show the potential of RNA interference as an effective postexposure treatment strategy for people infected with Ebola virus, and suggest that this strategy might also be useful for treatment of other emerging viral infections. FUNDING: Defense Threat Reduction Agency.
Asunto(s)
Ebolavirus/genética , Fiebre Hemorrágica Ebola/prevención & control , Interferencia de ARN , ARN Interferente Pequeño/uso terapéutico , Animales , Chlorocebus aethiops , Ebolavirus/aislamiento & purificación , Ebolavirus/fisiología , Femenino , Fiebre Hemorrágica Ebola/virología , Infusiones Intravenosas , Interferón-alfa/biosíntesis , Interleucina-6/biosíntesis , Macaca mulatta , Masculino , Ratones , Ratones Endogámicos ICR , ARN Interferente Pequeño/efectos adversos , ARN Interferente Pequeño/farmacología , Células Vero/virología , Proteínas Virales/genética , Viremia , Replicación ViralRESUMEN
TLR9 recognizes CpG motifs present in pathogenic DNA and triggers potent immune responses. It is generally accepted that TLR9 distinguishes pathogenic DNA based, in part, on methylation status, where TLR9 binds unmethylated but not methylated CpG. However, we showed that methylated CpG induces potent TLR9-mediated responses when delivered in lipid nanoparticles. In this article, we report that methylation dictates the ability of free CpG DNA to colocalize with TLR9 in late endosomes. However, when delivered in lipid nanoparticles, CpG DNA and TLR9 colocalize, regardless of methylation status. Therefore, it is proposed that the ability of immune cells to distinguish unmethylated pathogenic from methylated mammalian DNA is controlled by a mechanism that regulates TLR9 mobilization and colocalization rather than a differential binding affinity.
Asunto(s)
Metilación de ADN/inmunología , Endosomas/inmunología , Oligodesoxirribonucleótidos/metabolismo , Receptor Toll-Like 9/metabolismo , Animales , Separación Celular , Femenino , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Liposomas , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Microscopía Confocal , Nanopartículas , Oligodesoxirribonucleótidos/administración & dosificación , Oligodesoxirribonucleótidos/inmunología , Transporte de Proteínas/inmunología , Receptor Toll-Like 9/inmunologíaRESUMEN
Although it is well documented that the immunological activity of cytosine-guanine (CpG) motifs is abrogated by 5' methylation of the cytosine residue, encapsulation within stabilized lipid nanoparticles endows these methylated cytosine-guanine- (mCpG-) containing oligonucleotides (ODNs) with potent immunostimulatory activity in murine animal models. Surprisingly, not only do liposomal nanoparticulate (LN) mCpG ODN possess immunostimulatory activity, their potency is found to be equivalent and often greater than the equivalent unmethylated form, as judged by a number of ex vivo innate and adaptive immune parameters and anti-tumor efficacy in murine models. Preliminary data indicate that both methylated and unmethylated CpG ODN act through a common receptor signaling pathway, specifically via toll-like receptor (TLR) 9, based on observations of up-regulated TLR9 expression, induction of nitric oxide and dependence on endosomal maturation. This is confirmed in TLR9 knockout animals which show no immunostimulatory activity following treatment with LN-mCpG ODN. These data, therefore, indicate that the mCpG DNA is fully competent to interact with TLR9 to initiate potent immune responses. Furthermore, this work implicates an as yet unidentified mechanism upstream of TLR9 which regulates the relative activities of free methylated versus unmethylated CpG ODN that is effectively bypassed by particulate delivery of CpG ODN.
Asunto(s)
Adyuvantes Inmunológicos/administración & dosificación , Metilación de ADN , Nanopartículas/administración & dosificación , Oligodesoxirribonucleótidos/administración & dosificación , Receptor Toll-Like 9/efectos de los fármacos , Adyuvantes Inmunológicos/farmacología , Animales , Línea Celular Tumoral , Citocinas/sangre , Femenino , Inmunidad Activa , Inmunidad Innata , Liposomas , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Óxido Nítrico/inmunología , Óxido Nítrico/metabolismo , Oligodesoxirribonucleótidos/farmacología , Receptor Toll-Like 9/inmunología , Regulación hacia Arriba/inmunologíaRESUMEN
There has been significant interest in the potential of cytosine-guanine (CpG) containing oligodeoxynucleotides (ODN) as an immunotherapy for malignant, infectious and allergic diseases. While human trials have yielded promising results, clinical use of free CpG ODN still faces several challenges which limit their effectiveness. These include suboptimal in vivo stability, toxicity, unfavorable pharmacokinetic/biodistribution characteristics, lack of specificity for target cells and the requirement for intracellular uptake. To overcome these challenges, optimized lipid-based delivery systems have been developed to protect the CpG ODN payload, modify their circulation/distribution so as to enhance immune cell targeting and facilitate intracellular uptake. Ultimately, lipid-mediated delivery has the capacity to increase the immunopotency of CpG ODN and enhance their prophylactic or therapeutic efficacy in a range of diseases. Lipid-encapsulation provides a feasible strategy to optimize the immunostimulatory activity and immunotherapeutic efficacy of CpG ODN, thereby allowing their full clinical potential to be realized.
Asunto(s)
Adyuvantes Inmunológicos/administración & dosificación , Islas de CpG , Sistemas de Liberación de Medicamentos , Lípidos , Oligodesoxirribonucleótidos/administración & dosificación , Adyuvantes Inmunológicos/farmacocinética , Adyuvantes Inmunológicos/farmacología , Animales , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/inmunología , Composición de Medicamentos , Humanos , Inmunoterapia , Liposomas , Nanopartículas , Oligodesoxirribonucleótidos/farmacocinética , Oligodesoxirribonucleótidos/farmacología , Vacunas/administración & dosificación , Vacunas/uso terapéuticoRESUMEN
BACKGROUND: DNA vaccines offer unique potential for generating protective and therapeutic immunity against infectious and malignant diseases. Unfortunately, rapid degradation and poor cellular uptake has significantly limited the efficacy of 'naked' plasmid DNA vaccines. We have previously described stabilized plasmid lipid particles (SPLP) as effective nonviral gene delivery vehicles for the transfection of tumours at distal sites following intravenous administration. Based on their low toxicity and favourable transfection profile following systemic administration, we investigate SPLP as gene delivery vehicles for the generation of a systemically administered genetic vaccine. METHODS: The uptake of SPLP and their ability to transfect splenic antigen presenting cells (APC) following systemic administration is assessed through fluorescently-labelled SPLP in combination with phenotype markers and a very sensitive flow cytometry-based assay for the detection of the transgene, beta-galactosidase. The priming of antigen-specific adaptive and humoural immune responses following vaccination with SPLP alone or in combination with liposomal nanoparticle encapsulated CpG-ODN containing oligodeoxynucleotides (LN CpG-ODN) is characterized through the use of antigen-specific cytotoxicity assays, interferon-gamma secretion assays and enzyme-linked immunosorbant assay. RESULTS: We demonstrate that SPLP are taken up by and transfect APC in the spleen following intravenous administration and that, in the presence of a strong immunostimulatory signal provided by LN CpG-ODN, are able to prime transgene-specific humoural and cellular immune responses. CONCLUSIONS: SPLP represent an effective candidate for the nonviral delivery of a systemic genetic vaccine when combined with additional immune stimulation provided by LN CpG-ODN.
Asunto(s)
Liposomas/administración & dosificación , Nanopartículas/administración & dosificación , Oligodesoxirribonucleótidos/administración & dosificación , Vacunas de ADN/administración & dosificación , Animales , Células Presentadoras de Antígenos/metabolismo , Antígenos CD11/metabolismo , Células Cultivadas , Femenino , Terapia Genética , Vectores Genéticos/administración & dosificación , Lípidos/química , Ratones , Ratones Endogámicos BALB C , TransfecciónRESUMEN
The encapsulation of conventional drugs in lipid nanoparticles (LNs) has been extensively utilized to enhance therapeutic activity by altering their pharmacokinetic (PK) and biodistribution (BD) properties. We have previously shown that the immunostimulatory activity of unmethylated cytidine-guanosine (CpG)-containing immunostimulatory oligodeoxynucleotides (ODN) is greatly enhanced when encapsulated in an LN (LN CpG-ODN). Here, we investigate the effect of circulation lifetime (determined by lipid composition) and drug-to-lipid (D/L) ratio of intravenously (i.v.) administered LN CpG-ODN on PK, BD, and cellular uptake and correlate these parameters with the immunostimulatory activity. Results from these studies show that despite significant differences in the circulation lifetime and the D/L ratio, the immune response is similar with respect to immune cell activation and cytolytic activity in the spleen and the blood compartments. Our findings indicate that the benefits of liposomal nanoparticles for the delivery of immunomodulatory drugs such as CpG-ODN are defined by a different paradigm than that for conventional drugs.
Asunto(s)
Islas de CpG , Factores Inmunológicos/administración & dosificación , Lípidos/administración & dosificación , Nanopartículas , Oligodesoxirribonucleótidos/administración & dosificación , Animales , Secuencia de Bases , Femenino , Factores Inmunológicos/sangre , Factores Inmunológicos/farmacocinética , Factores Inmunológicos/farmacología , Infusiones Intravenosas , Ratones , Ratones Endogámicos , Oligodesoxirribonucleótidos/sangre , Oligodesoxirribonucleótidos/farmacocinética , Oligodesoxirribonucleótidos/farmacología , Distribución TisularRESUMEN
Immunostimulatory oligodeoxynucleotides (ODN) containing cytosine-guanine (CpG) motifs are powerful stimulators of innate as well as adaptive immune responses, exerting their activity through triggering of the Toll-like receptor 9. We have previously shown that encapsulation in liposomal nanoparticles (LN) enhances the immunostimulatory activity of CpG ODN (LN-CpG ODN) (Mui et al. in J Pharmacol Exp Ther 298:1185, 2001). In this work we investigate the effect of encapsulation on the immunopotency of subcutaneously (s.c.) administered CpG ODN with regard to activation of innate immune cells as well as its ability to act as a vaccine adjuvant with tumor-associated antigens (TAAs) to induce antigen (Ag)-specific, adaptive responses and anti-tumor activity in murine models. It is shown that encapsulation specifically targets CpG ODN for uptake by immune cells. This may provide the basis, at least in part, for the significantly enhanced immunostimulatory activity of LN-CpG ODN, inducing potent innate (as judged by immune cell activation and plasma cytokine/chemokine levels) and adaptive, Ag-specific (as judged by MHC tetramer positive T lymphocytes, IFN-gamma secretion and cytotoxicity) immune responses. Finally, in efficacy studies, it is shown that liposomal encapsulation enhances the ability of CpG ODN to adjuvanate adaptive immune responses against co-administered TAAs after s.c. immunization, inducing effective anti-tumor activity against both model and syngeneic tumor Ags in murine tumor models of thymoma and melanoma.