RESUMO
Primary hyperoxalurias (PHs) are autosomal recessive disorders caused by the overproduction of oxalate leading to calcium oxalate precipitation in the kidney and eventually to end-stage renal disease. One promising strategy to treat PHs is to reduce the hepatic production of oxalate through substrate reduction therapy by inhibiting liver-specific glycolate oxidase (GO), which controls the conversion of glycolate to glyoxylate, the proposed main precursor to oxalate. Alternatively, diminishing the amount of hepatic lactate dehydrogenase (LDH) expression, the proposed key enzyme responsible for converting glyoxylate to oxalate, should directly prevent the accumulation of oxalate in PH patients. Using RNAi, we provide the first in vivo evidence in mammals to support LDH as the key enzyme responsible for converting glyoxylate to oxalate. In addition, we demonstrate that reduction of hepatic LDH achieves efficient oxalate reduction and prevents calcium oxalate crystal deposition in genetically engineered mouse models of PH types 1 (PH1) and 2 (PH2), as well as in chemically induced PH mouse models. Repression of hepatic LDH in mice did not cause any acute elevation of circulating liver enzymes, lactate acidosis, or exertional myopathy, suggesting further evaluation of liver-specific inhibition of LDH as a potential approach for treating PH1 and PH2 is warranted.
Assuntos
Hiperoxalúria Primária/terapia , L-Lactato Desidrogenase/antagonistas & inibidores , Oxalatos/metabolismo , Interferência de RNA/fisiologia , Animais , Modelos Animais de Doenças , Inativação Gênica , Humanos , Hiperoxalúria Primária/genética , Hiperoxalúria Primária/metabolismo , L-Lactato Desidrogenase/genética , Fígado/enzimologia , CamundongosRESUMO
Primary hyperoxaluria type 1 (PH1) is an autosomal recessive, metabolic disorder caused by mutations of alanine-glyoxylate aminotransferase (AGT), a key hepatic enzyme in the detoxification of glyoxylate arising from multiple normal metabolic pathways to glycine. Accumulation of glyoxylate, a precursor of oxalate, leads to the overproduction of oxalate in the liver, which accumulates to high levels in kidneys and urine. Crystalization of calcium oxalate (CaOx) in the kidney ultimately results in renal failure. Currently, the only treatment effective in reduction of oxalate production in patients who do not respond to high-dose vitamin B6 therapy is a combined liver/kidney transplant. We explored an alternative approach to prevent glyoxylate production using Dicer-substrate small interfering RNAs (DsiRNAs) targeting hydroxyacid oxidase 1 (HAO1) mRNA which encodes glycolate oxidase (GO), to reduce the hepatic conversion of glycolate to glyoxylate. This approach efficiently reduces GO mRNA and protein in the livers of mice and nonhuman primates. Reduction of hepatic GO leads to normalization of urine oxalate levels and reduces CaOx deposition in a preclinical mouse model of PH1. Our results support the use of DsiRNA to reduce liver GO levels as a potential therapeutic approach to treat PH1.
Assuntos
Oxirredutases do Álcool/genética , Oxalato de Cálcio/metabolismo , Hiperoxalúria Primária/terapia , RNA Interferente Pequeno/administração & dosagem , Animais , RNA Helicases DEAD-box/metabolismo , Modelos Animais de Doenças , Glioxilatos/urina , Humanos , Hiperoxalúria Primária/enzimologia , Hiperoxalúria Primária/urina , Fígado/metabolismo , Camundongos , Nanopartículas/química , RNA Interferente Pequeno/farmacologia , Ribonuclease III/metabolismoRESUMO
SREBP cleavage-activating protein (SCAP) is a key protein in the regulation of lipid metabolism and a potential target for treatment of dyslipidemia. SCAP is required for activation of the transcription factors SREBP-1 and -2. SREBPs regulate the expression of genes involved in fatty acid and cholesterol biosynthesis, and LDL-C clearance through the regulation of LDL receptor (LDLR) and PCSK9 expression. To further test the potential of SCAP as a novel target for treatment of dyslipidemia, we used siRNAs to inhibit hepatic SCAP expression and assess the effect on PCSK9, LDLR, and lipids in mice and rhesus monkeys. In mice, robust liver Scap mRNA knockdown (KD) was achieved, accompanied by dose-dependent reduction in SREBP-regulated gene expression, de novo lipogenesis, and plasma PCSK9 and lipids. In rhesus monkeys, over 90% SCAP mRNA KD was achieved resulting in approximately 75, 50, and 50% reduction of plasma PCSK9, TG, and LDL-C, respectively. Inhibition of SCAP function was demonstrated by reduced expression of SREBP-regulated genes and de novo lipogenesis. In conclusion, siRNA-mediated inhibition of SCAP resulted in a significant reduction in circulating PCSK9 and LDL-C in rodent and primate models supporting SCAP as a novel target for the treatment of dyslipidemia.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/genética , Lipídeos/sangue , Proteínas de Membrana/genética , Pró-Proteína Convertase 9/genética , RNA Interferente Pequeno/genética , Receptores de LDL/genética , Animais , Feminino , Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Hipolipemiantes/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lipogênese , Fígado/enzimologia , Macaca mulatta , Masculino , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Pró-Proteína Convertase 9/metabolismo , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de LDL/metabolismo , Transdução de Sinais , Sinvastatina/farmacologia , Proteínas de Ligação a Elemento Regulador de Esterol/genética , Proteínas de Ligação a Elemento Regulador de Esterol/metabolismoRESUMO
European bat lyssaviruses type 1 (EBLV-1) and type 2 (EBLV-2) circulate within bat populations throughout Europe and are capable of causing disease indistinguishable from that caused by classical rabies virus (RABV). However, the determinants of viral fitness and pathogenicity are poorly understood. Full-length genome clones based on the highly attenuated, non-neuroinvasive, RABV vaccine strain (SAD-B19) were constructed with the glycoprotein (G) of either SAD-B19 (SN), of EBLV-1 (SN-1) or EBLV-2 (SN-2). In vitro characterization of SN-1 and SN-2 in comparison to wild-type EBLVs demonstrated that the substitution of G affected the final virus titre and antigenicity. In vivo, following peripheral infection with a high viral dose (10(4) f.f.u.), animals infected with SN-1 had reduced survivorship relative to infection with SN, resulting in survivorship similar to animals infected with EBLV-1. The histopathological changes and antigen distribution observed for SN-1 were more representative of those observed with SN than with EBLV-1. EBLV-2 was unable to achieve a titre equivalent to that of the other viruses. Therefore, a reduced-dose experiment (10(3) f.f.u.) was undertaken in vivo to compare EBLV-2 and SN-2, which resulted in 100â% survivorship for all recombinant viruses (SN, SN-1 and SN-2) while clinical disease developed in mice infected with the EBLVs. These data indicate that interspecies replacement of G has an effect on virus titre in vitro, probably as a result of suboptimal G-matrix protein interactions, and influences the survival outcome following a peripheral challenge with a high virus titre in mice.
Assuntos
Glicoproteínas/metabolismo , Lyssavirus/genética , Lyssavirus/patogenicidade , Proteínas Virais/metabolismo , Fatores de Virulência/metabolismo , Animais , Antígenos Virais/genética , Antígenos Virais/imunologia , Antígenos Virais/metabolismo , Encéfalo/patologia , Encéfalo/virologia , Modelos Animais de Doenças , Glicoproteínas/genética , Glicoproteínas/imunologia , Histocitoquímica , Imuno-Histoquímica , Lyssavirus/imunologia , Camundongos , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Recombinação Genética , Infecções por Rhabdoviridae/patologia , Infecções por Rhabdoviridae/virologia , Análise de Sobrevida , Carga Viral , Proteínas Virais/genética , Proteínas Virais/imunologia , Fatores de Virulência/genética , Fatores de Virulência/imunologiaRESUMO
Effective small interfering RNA (siRNA)-mediated therapeutics require the siRNA to be delivered into the cellular RNA-induced silencing complex (RISC). Quantitative information of this essential delivery step is currently inferred from the efficacy of gene silencing and siRNA uptake in the tissue. Here we report an approach to directly quantify siRNA in the RISC in rodents and monkey. This is achieved by specific immunoprecipitation of the RISC from tissue lysates and quantification of small RNAs in the immunoprecipitates by stem-loop PCR. The method, expected to be independent of delivery vehicle and target, is label-free, and the throughput is acceptable for preclinical animal studies. We characterized a lipid-formulated siRNA by integrating these approaches and obtained a quantitative perspective on siRNA tissue accumulation, RISC loading, and gene silencing. The described methodologies have utility for the study of silencing mechanism, the development of siRNA therapeutics, and clinical trial design.
Assuntos
Técnicas de Transferência de Genes , RNA Interferente Pequeno/genética , Animais , Animais Geneticamente Modificados , Anticorpos/isolamento & purificação , Anticorpos/metabolismo , Anticorpos/farmacologia , Especificidade de Anticorpos , Proteínas Argonautas , Células Cultivadas , Fator de Iniciação 2 em Eucariotos/imunologia , Fator de Iniciação 2 em Eucariotos/metabolismo , Estudos de Avaliação como Assunto , Feminino , Inativação Gênica/fisiologia , Marcação de Genes/métodos , Técnicas de Transferência de Genes/normas , Humanos , Imunoprecipitação/métodos , Imunoprecipitação/normas , Macaca mulatta , Camundongos , Camundongos Endogâmicos ICR , Ligação Proteica , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Sprague-Dawley , RoedoresRESUMO
Despite recent progress, systemic delivery remains the major hurdle for development of safe and effective small inhibitory RNA (siRNA)-based therapeutics. Encapsulation of siRNA into liposomes is a promising option to overcome obstacles such as low stability in serum and inefficient internalization by target cells. However, a major liability of liposomes is the potential to induce an acute inflammatory response, thereby increasing the risk of numerous adverse effects. In this study, we characterized a liposomal siRNA delivery vehicle, LNP201, which is capable of silencing an mRNA target in mouse liver by over 80%. The biodistribution profile, efficacy after single and multiple doses, mechanism of action, and inflammatory toxicity are characterized for LNP201. Furthermore, we demonstrate that the glucocorticoid receptor (GR) agonist dexamethasone (Dex) inhibits LNP201-induced cytokine release, inflammatory gene induction, and mitogen-activated protein kinase (MAPK) phosphorylation in multiple tissues. These data present a possible clinical strategy for increasing the safety profile of siRNA-based drugs while maintaining the potency of gene silencing.
Assuntos
Dexametasona/uso terapêutico , Nanopartículas/efeitos adversos , RNA Interferente Pequeno/imunologia , RNA Interferente Pequeno/metabolismo , Animais , Feminino , Inativação Gênica , Inflamação/induzido quimicamente , Inflamação/tratamento farmacológico , Camundongos , Nanopartículas/administração & dosagem , RNA Interferente Pequeno/administração & dosagem , Receptores de Glucocorticoides/agonistasRESUMO
Colorectal carcinomas harbor well-defined genetic abnormalities, including aberrant activation of Wnt/ß-catenin and MAPK pathways, often simultaneously. Although the MAPK pathway can be targeted using potent small-molecule drugs, including BRAF and MEK inhibitors, ß-catenin inhibition has been historically challenging. RNAi approaches have advanced to the stage of clinical viability and are especially well suited for transcriptional modulators, such as ß-catenin. In this study, we report therapeutic effects of combined targeting of these pathways with pharmacologic agents. Using a recently described tumor-selective nanoparticle containing a ß-catenin-targeting RNAi trigger, in combination with the FDA-approved MEK inhibitor (MEKi) trametinib, we demonstrate synergistic tumor growth inhibition in in vivo models of colorectal cancer, melanoma, and hepatocellular carcinoma. At dose levels that were insufficient to significantly impact tumor growth as monotherapies, combination regimens resulted in synergistic efficacy and complete tumor growth inhibition. Importantly, dual MEKi/RNAi therapy dramatically improved survival of mice bearing colorectal cancer liver metastases. In addition, pharmacologic silencing of ß-catenin mRNA was effective against tumors that are inherently resistant or that acquire drug-induced resistance to trametinib. These results provide a strong rationale for clinical evaluation of this dual-targeting approach for cancers harboring Wnt/ß-catenin and MAPK pathway mutations. Mol Cancer Ther; 17(2); 544-53. ©2017 AACR.
Assuntos
Neoplasias Colorretais/terapia , MAP Quinase Quinase Quinases/antagonistas & inibidores , Piridonas/farmacologia , Pirimidinonas/farmacologia , RNA Mensageiro/genética , RNA Interferente Pequeno/administração & dosagem , beta Catenina/genética , Animais , Linhagem Celular Tumoral , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Sinergismo Farmacológico , Inativação Gênica , Xenoenxertos , Humanos , Neoplasias Hepáticas Experimentais/secundário , MAP Quinase Quinase Quinases/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Camundongos Nus , Nanopartículas/administração & dosagem , Inibidores de Proteínas Quinases/farmacologia , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , Via de Sinalização Wnt/efeitos dos fármacos , beta Catenina/metabolismoRESUMO
The Wnt/ß-catenin pathway is among the most frequently altered signaling networks in human cancers. Despite decades of preclinical and clinical research, efficient therapeutic targeting of Wnt/ß-catenin has been elusive. RNA interference (RNAi) technology silences genes at the mRNA level and therefore can be applied to previously undruggable targets. Lipid nanoparticles (LNP) represent an elegant solution for the delivery of RNAi-triggering oligonucleotides to disease-relevant tissues, but have been mostly restricted to applications in the liver. In this study, we systematically tuned the composition of a prototype LNP to enable tumor-selective delivery of a Dicer-substrate siRNA (DsiRNA) targeting CTNNB1, the gene encoding ß-catenin. This formulation, termed EnCore-R, demonstrated pharmacodynamic activity in subcutaneous human tumor xenografts, orthotopic patient-derived xenograft (PDX) tumors, disseminated hematopoietic tumors, genetically induced primary liver tumors, metastatic colorectal tumors, and murine metastatic melanoma. DsiRNA delivery was homogeneous in tumor sections, selective over normal liver and independent of apolipoprotein-E binding. Significant tumor growth inhibition was achieved in Wnt-dependent colorectal and hepatocellular carcinoma models, but not in Wnt-independent tumors. Finally, no evidence of accelerated blood clearance or sustained liver transaminase elevation was observed after repeated dosing in nonhuman primates. These data support further investigation to gain mechanistic insight, optimize dose regimens, and identify efficacious combinations with standard-of-care therapeutics. Mol Cancer Ther; 15(9); 2143-54. ©2016 AACR.
Assuntos
Neoplasias/genética , Interferência de RNA , RNA Interferente Pequeno/genética , beta Catenina/genética , Animais , Apolipoproteínas E/química , Apolipoproteínas E/metabolismo , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Modelos Animais de Doenças , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Humanos , Lipídeos/química , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Masculino , Melanoma Experimental , Camundongos , Nanopartículas/química , Metástase Neoplásica , Neoplasias/metabolismo , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/química , Relação Estrutura-Atividade , Via de Sinalização Wnt , Ensaios Antitumorais Modelo de Xenoenxerto , beta Catenina/metabolismo , Proteínas ras/genética , Proteínas ras/metabolismoRESUMO
Decreased production of erythropoietin (EPO) causes anemia in patients with chronic kidney disease, and recombinant human EPO is used to treat renal failure associated anemia. The liver, the main EPO-producing organ in utero, maintains the capacity to produce EPO in the adult but in insufficient quantities to restore hemoglobin levels to normal in patients with impaired renal function. Inhibition of prolyl-4-hydroxylase domain (PHD) proteins is known to cause an increase in EPO production through its effects on hypoxia inducible factor. Here, we utilized small interfering RNA (siRNA) targeting EGLN1, the gene encoding the PHD2 protein, to investigate the phenotypic consequences in nonhuman primates. A single, well-tolerated intravenous dose of an optimized EGLN1 siRNA encapsulated in a lipid nanoparticle formulation caused robust mRNA silencing in the liver, leading to increases in serum EPO and hemoglobin. The siRNA-induced erythropoiesis was dose-dependent and was sustained for at least 2 months. These data point to the potential for an RNA interference-based, liver-targeted therapeutic approach for the treatment of anemia.
Assuntos
Eritropoese/efeitos dos fármacos , Prolina Dioxigenases do Fator Induzível por Hipóxia/antagonistas & inibidores , Interferência de RNA , RNA Interferente Pequeno/administração & dosagem , Animais , Eritropoetina/metabolismo , Prolina Dioxigenases do Fator Induzível por Hipóxia/genética , Fígado/efeitos dos fármacos , Macaca mulattaRESUMO
INTRODUCTION: Quantitative pharmacokinetic measurement of short nucleotide sequences in animal tissues is critical to the successful development of siRNA-based drugs. Stem-loop qRT-PCR is a sensitive and precise methodology, but the effect of biological matrix and purity of the input sample has yet to be investigated. RESULTS: The impact of lipid encapsulation, siRNA chemical modification and purity of the biological matrix on the stem-loop qRT-PCR assay was investigated. A comparison of siRNA standard curves in mouse liver homogenates before and after isolation of total RNA uncovered the potential for erroneous measurement due to significant loss of siRNA on purification columns. Recovery of chemically stabilized siRNA was improved by omission of the DNAse I digestion during RNA isolation. The stem-loop qRT-PCR method demonstrated excellent sensitivity and efficiency in mouse liver homogenates, plasma and whole blood. An optimized protocol based on these findings was used to quantitate siRNA in tissues after dosing mice with two different lipid nanoparticle formulations containing siRNA payloads. CONCLUSIONS: Assay of crude homogenates, whole blood or plasma is more accurate, less resource intensive and more amenable to clinical translation than measurement of column-purified total RNA.
Assuntos
RNA Interferente Pequeno/isolamento & purificação , RNA Interferente Pequeno/farmacocinética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Animais , Química Farmacêutica , Estudos de Avaliação como Assunto , Feminino , Lipossomos/administração & dosagem , Lipossomos/química , Camundongos , Nanopartículas/administração & dosagem , Nanopartículas/química , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/químicaRESUMO
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.
Assuntos
Produtos do Gene env/imunologia , Produtos do Gene gag/imunologia , Vacinas contra a SAIDS/uso terapêutico , Síndrome de Imunodeficiência Adquirida dos Símios/prevenção & controle , Vacinas Sintéticas/uso terapêutico , Animais , Primers do DNA , Produtos do Gene env/genética , Produtos do Gene gag/genética , Vetores Genéticos , Macaca mulatta , Masculino , RNA Viral/análise , Vírus da Raiva/genética , Vírus da Imunodeficiência Símia/genéticaRESUMO
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.
Assuntos
Vacinas contra Antraz/administração & dosagem , Antraz/prevenção & controle , Antígenos de Bactérias/genética , Antígenos Virais/genética , Bacillus anthracis/imunologia , Toxinas Bacterianas/genética , Vetores Genéticos/genética , Glicoproteínas/genética , Vacina Antirrábica/genética , Vacinação , Proteínas do Envelope Viral/genética , Animais , Antraz/sangue , Antraz/imunologia , Anticorpos Antibacterianos/sangue , Anticorpos Antivirais/sangue , Especificidade de Anticorpos , Antígenos de Bactérias/imunologia , Antígenos Virais/imunologia , Toxinas Bacterianas/imunologia , Bioterrorismo/prevenção & controle , Vetores Genéticos/imunologia , Glicoproteínas/imunologia , Esquemas de Imunização , Injeções Intramusculares , Contagem de Linfócitos , Linfócitos/citologia , Linfócitos/imunologia , Camundongos , Vacina Antirrábica/imunologia , Recombinação Genética , Células Th2/imunologia , Vacinas Sintéticas/administração & dosagem , Proteínas do Envelope Viral/imunologiaRESUMO
Recombinant rabies virus (RV) vaccine strain-based vectors expressing HIV-1 antigens have been shown to induce strong and long-lasting cellular but modest humoral responses against the expressed antigens in mice. However, an effective vaccine against HIV-1 may require stronger responses, and the development of such an immune response may depend on the presence of certain cytokines at the time of the inoculation. Here, we describe several new RV-based vaccine vehicles expressing HIV-1 Gag or envelope (Env) and murine IL-2 or IL-4. Cells infected with recombinant RVs expressed high levels of functional IL-2 or IL-4 in culture supernatants in addition to HIV-1 proteins. The recombinant RV expressing IL-4 was highly attenuated in a cytokine-independent manner, indicating that the insertion of two foreign genes into the RV genome is mainly responsible for the attenuation observed. The expression of IL-4 resulted in a decrease in the cellular immune response against HIV-1 Gag and Env when compared with the parental virus not expressing IL-4 and only 2 of 20 mice seroconverted to HIV-1 Env after two inoculations. The IL-2-expressing RV was completely apathogenic after direct intracranial inoculation of mice. In addition, mice immunized with IL-2 maintained strong anti-HIV-1 Gag and Env cellular responses and consistently induced seroconversion against HIV-1 Env after two inoculations. This suggests the potential use of IL-2 in RV-based HIV-1 vaccine strategies, which may require the induction of both arms of the immune response.
Assuntos
Vacinas contra a AIDS/imunologia , Antígenos HIV/imunologia , HIV-1/imunologia , Interleucina-2/imunologia , Rhabdoviridae/genética , Vacinas contra a AIDS/genética , Animais , Linhagem Celular , Proliferação de Células , Feminino , Produtos do Gene env/genética , Produtos do Gene env/imunologia , Produtos do Gene gag/genética , Produtos do Gene gag/imunologia , Vetores Genéticos/genética , Antígenos HIV/genética , HIV-1/genética , Interleucina-2/genética , Interleucina-4/imunologia , Camundongos , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologiaRESUMO
Recombinant rhabdovirus vectors expressing human immunodeficiency virus (HIV) and/or simian immunodeficiency virus (SIV) proteins have been shown to induce strong immune responses in mice and rhesus macaques. However, the finding that such responses protect rhesus macaques from AIDS-like disease but not from infection indicates that further improvements for these vectors are needed. Here, we designed a prime-boost schedule consisting of a rabies virus (RV) vaccine strain and a recombinant vesicular stomatitis virus (VSV) both expressing HIV Envelope (Env). Mice were primed and boosted with the two vaccine vehicles by different routes and in different combinations. Mucosal and systemic humoral responses were assessed using enzyme linked immunosorbent assay (ELISA) while the cellular immune response was determined by an IFN-gamma ELISPOT assay. We found that an immunization combination of RV and VSV elicited the highest titers of anti-Env antibodies and the greatest amount of Env-specific IFN-gamma secreting cells pre- and post-challenge with a recombinant vaccinia virus expressing HIV(89.6) Env. Furthermore, intramuscular immunization did not induce antigen-specific mucosal antibodies while intranasal inoculation stimulated vector-specific IgA antibodies in vaginal washings and serum. Our results show that it is feasible to elicit robust cellular and humoral anti-HIV responses using two different live attenuated Rhabdovirus vectors to sequentially prime and boost.
Assuntos
Produtos do Gene env/imunologia , HIV-1/imunologia , Rhabdoviridae/fisiologia , Vacinas contra a AIDS/imunologia , Animais , Ensaio de Imunoadsorção Enzimática , Feminino , Vetores Genéticos/fisiologia , Anticorpos Anti-HIV/biossíntese , Imunidade Celular , Imunidade nas Mucosas , Esquemas de Imunização , Interferon gama/sangue , Camundongos , Camundongos Endogâmicos BALB C , Vírus da Raiva/imunologia , Vacinas Sintéticas/imunologia , Vírus da Estomatite Vesicular Indiana/fisiologiaRESUMO
Recombinant rabies virus (RV) vaccine strain-based vectors have been successfully developed as vaccines against other viral diseases (J. P. McGettigan et al., J. Virol. 75:4430-4434, 2001; McGettigan et al., J. Virol. 75:8724-8732, 2001; C. A. Siler et al., Virology 292:24-34, 2002), and safety concerns have recently been addressed (McGettigan et al., J. Virol. 77:237-244, 2003). However, size limitations of the vectors may restrict their use for development of vaccine applications that require the expression of large and multiple foreign antigens. Here we describe a new RV-based vaccine vehicle expressing 4.4 kb of the human immunodeficiency virus type 1 (HIV-1) Gag-Pol precursor Pr160. Our results indicate that Pr160 is expressed and processed, as demonstrated by immunostaining and Western blotting. Electron microscopy studies showed both immature and mature HIV-1 virus-like particles (VLPs), indicating that the expressed HIV-1 Gag Pr55 precursor was processed properly by the HIV-1 protease. A functional assay also confirmed the cleavage and functional expression of the HIV-1 reverse transcriptase (RT) from the modified RV genome. In the next step, we constructed and recovered a new RV vaccine strain-based vector expressing a chimeric HIV-1(89.6P) RV envelope protein from an additional RV transcription unit located between the RV nucleoprotein (N) and phosphoprotein (P) in addition to HIV-1 Pr160. The 2.2-kb chimeric HIV-1/RV envelope protein is composed of the HIV-1 Env ectodomain (ED) and transmembrane domain (TD) fused to RV glycoprotein (G) cytoplasmic domain (CD), which is required for efficient incorporation of HIV-1 Env into RV particles. Of note, the expression of both HIV-1 Env and HIV-1 Pr160 resulted in an increase in the rhabdoviral genome of >55%. Both rhabdovirus-expressed HIV-1 precursor proteins were functional, as indicated by RT activity and Env-based fusion assays. These findings demonstrate that both multiple and very large foreign genes can be effectively expressed by RV-based vectors. This research opens up the possibility for the further improvement of rhabdovirus-based HIV-1 vaccines and their use to express large foreign proteins, perhaps from multiple human pathogens.
Assuntos
Vacinas contra a AIDS/imunologia , Proteínas de Fusão gag-pol/imunologia , HIV-1/imunologia , Rhabdoviridae/genética , Vacinas Sintéticas/imunologia , Proteínas do Envelope Viral/imunologia , Proteínas de Fusão gag-pol/genética , Vetores Genéticos , Células HeLa , Humanos , Precursores de Proteínas/imunologia , Proteínas Recombinantes/imunologia , Linfócitos T Citotóxicos/imunologia , Vírion/fisiologiaRESUMO
Rabies virus (RV) nucleoprotein (N) tightly encapsidates the genomic and antigenomic RNA of RV to form the viral ribonucleoprotein (RNP) complex. Antigens, such as N, presented in a highly organized structure are sufficient and even desirable to activate B cells to proliferate and produce antibodies. In addition to activating B cells to proliferate, it has been shown that RV N in the RNP complex induces potent T helper cell responses resulting in long-lasting and strong humoral immune responses against RV. The possibility to systematically incorporate foreign genes into the genome of RV and produce a recombinant virus allows us to examine whether the immunogenicity of foreign antigens can be enhanced by incorporation into the RV RNP structure. To test this hypothesis we constructed a recombinant RV expressing a RV N-GFP fusion protein. The chimeric N-GFP fusion protein was efficiently expressed and incorporated into RV RNP and virions. Moreover, the recombinant RNP induces a strong humoral immune response against GFP in mice. In contrast, mice inoculated with GFP alone or a combination of wild-type RV RNPs and GFP did not trigger any GFP-specific humoral responses using the same immunization schedule. These data indicate the usefulness of RV-based vectors as killed vaccines against other infectious diseases.