RESUMO
The COVID-19 pandemic continues to have devastating consequences on health and economy, even after the approval of safe and effective vaccines. Waning immunity, the emergence of variants of concern, breakthrough infections, and lack of global vaccine access and acceptance perpetuate the epidemic. Here, we demonstrate that a single injection of an adenoassociated virus (AAV)-based COVID-19 vaccine elicits at least 17-month-long neutralizing antibody responses in non-human primates at levels that were previously shown to protect from viral challenge. To improve the scalability of this durable vaccine candidate, we further optimized the vector design for greater potency at a reduced dose in mice and non-human primates. Finally, we show that the platform can be rapidly adapted to other variants of concern to robustly maintain immunogenicity and protect from challenge. In summary, we demonstrate this class of AAV can provide durable immunogenicity, provide protection at dose that is low and scalable, and be adapted readily to novel emerging vaccine antigens thus may provide a potent tool in the ongoing fight against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2).
Assuntos
COVID-19 , Vacinas Virais , Animais , Anticorpos Neutralizantes , Anticorpos Antivirais , COVID-19/prevenção & controle , Vacinas contra COVID-19 , Dependovirus/genética , Humanos , Macaca , Camundongos , Pandemias/prevenção & controle , SARS-CoV-2/genéticaRESUMO
Gene therapy provides a significant opportunity to treat a variety of inherited and acquired diseases. However, adverse immune responses toward the adeno-associated virus (AAV) antigens may limit its success. The mechanisms responsible for immunity or tolerance toward AAV-encoded transgene products remain poorly defined. Studies in mice demonstrate that AAV2/8 gene transfer to liver is associated with immunological hyporesponsiveness toward both AAV vector and antigenic transgene product. To evaluate the role of activation-induced cell death (AICD) and cytokine withdrawal (intrinsic cell death) in the deletion of mature T lymphocytes, we compared immunological responses in hepatic AAV2/8 transfer in murine recipients lacking the Fas receptor, and recipients overexpressing Bcl-xL, to WT murine counterparts. Prolonged transgene expression was dependent on both Fas signaling and Bcl-xL-regulated apoptosis in T cells. Abrogation of intrinsic cell death enhanced Th1 responses, whereas AICD functioned to limit neutralizing antibody production toward AAV2/8. In addition, immune hyporesponsiveness and stable transgene expression was dependent on upregulation of FasL expression on transduced hepatocytes and a corresponding apoptosis of infiltrating Fas (+) cells. These data provide evidence that both AICD and apoptosis due to cytokine withdrawal of lymphocytes are essential for immune hyporesponsiveness toward hepatic AAV2/8-encoded transgene product in the setting of liver gene transfer.
Assuntos
Apoptose , Citocinas/imunologia , Dependovirus/genética , Dependovirus/imunologia , Técnicas de Transferência de Genes , Fígado/imunologia , Proteína bcl-X/metabolismo , Receptor fas/metabolismo , Animais , Terapia Genética , Vetores Genéticos , Tolerância Imunológica , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Linfócitos T/imunologia , Proteína bcl-X/genéticaRESUMO
Activation of T cells to the capsid of adeno-associated virus (AAV) serotype 2 vectors has been implicated in liver toxicity in a recent human gene therapy trial of hemophilia B. To further investigate this kind of toxicity, we evaluated T-cell responses to AAV capsids after intramuscular injection of vectors into mice and nonhuman primates. High levels of T cells specific to capsids of vectors based on AAV2 and a phylogenetically related AAV variant were detected. Vectors from other AAV clades such as AAV8 (ref. 3), however, did not lead to activation of capsid-specific T cells. Through the generation of AAV2-AAV8 hybrids and the creation of site-directed mutations, we mapped the domain that directs the activation of T cells to the RXXR motif on VP3, which was previously shown to confer binding of the virion to heparan sulfate proteoglycan (HSPG). Evaluation of natural and engineered AAV variants showed direct correlations between heparin binding, uptake into human dendritic cells (DCs) and activation of capsid-specific T cells. The role of heparin binding in the activation of CD8(+) T cells may be useful in modulating the immunogenicity of antigens and improving the safety profile of existing AAV vectors for gene therapy.
Assuntos
Capsídeo/metabolismo , Dependovirus/genética , Vetores Genéticos , Heparina/metabolismo , Linfócitos T/metabolismo , Animais , Células CHO , Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , Cricetinae , Células Dendríticas/metabolismo , Dependovirus/classificação , Dependovirus/metabolismo , Marcadores Genéticos , Vetores Genéticos/administração & dosagem , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Células HeLa , Heparina/farmacologia , Humanos , Interferon gama/análise , Interferon gama/imunologia , Interleucina-4/farmacologia , Cinética , Leucócitos Mononucleares/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Macaca fascicularis , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Estrutura Terciária de Proteína , Sorotipagem , Fatores de TempoRESUMO
Although restoration of dystrophin expression via exon skipping in both cardiac and skeletal muscle has been successfully demonstrated in the mdx mouse, restoration of cardiac dystrophin expression in large animal models of Duchenne muscular dystrophy (DMD) has proven to be a challenge. In large animals, investigators have focused on using intravenous injection of antisense oligonucleotides (AO) to mediate exon skipping. In this study, we sought to optimize restoration of cardiac dystrophin expression in the golden retriever muscular dystrophy (GRMD) model using percutaneous transendocardial delivery of recombinant AAV6 (rAAV6) to deliver a modified U7 small nuclear RNA (snRNA) carrying antisense sequence to target the exon splicing enhancers of exons 6 and 8 and correct the disrupted reading frame. We demonstrate restoration of cardiac dystrophin expression at 13 months confirmed by reverse transcription-PCR (RT-PCR) and immunoblot as well as membrane localization by immunohistochemistry. This was accompanied by improved cardiac function as assessed by cardiac magnetic resonance imaging (MRI). Percutaneous transendocardial delivery of rAAV6 expressing a modified U7 exon skipping construct is a safe, effective method for restoration of dystrophin expression and improvement of cardiac function in the GRMD canine and may be easily translatable to human DMD patients.
Assuntos
Processamento Alternativo , Dependovirus/genética , Distrofina/genética , Vetores Genéticos/genética , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapia , Animais , Linhagem Celular , Modelos Animais de Doenças , Cães , Distrofina/metabolismo , Ecocardiografia , Éxons , Fibrose , Expressão Gênica , Ordem dos Genes , Técnicas de Transferência de Genes , Vetores Genéticos/farmacocinética , Genoma Viral , Humanos , Imageamento por Ressonância Magnética , Distrofia Muscular de Duchenne/diagnóstico , Miocárdio/patologia , RNA Mensageiro/metabolismoRESUMO
The adeno-associated viral vector (AAV) provides a safe and efficient gene therapy platform with several approved products that have marked therapeutic impact for patients. However, a major bottleneck in the development and commercialization of AAV remains the efficiency, cost, and scalability of AAV production. Chromatographic methods have the potential to allow purification at increased scales and lower cost but often require optimization specific to each serotype. Here, we demonstrate that the POROS CaptureSelect AAVX affinity resin efficiently captures a panel of 15 divergent AAV serotypes, including the commonly used AAV2, AAV8, AAV9, PHP.B, and Anc80. We also find that AAVX resin can be regenerated repeatedly without loss of efficiency or carry-over contamination. While AAV preps purified with AAVX showed a higher fraction of empty capsids than preps purified using iodixanol ultracentrifugation, the potency of the AAVX purified vectors was comparable with that of iodixanol purified vectors both in vitro and in vivo. Finally, optimization of the purification protocol resulted in a process with an overall efficiency of 65%-80% across all scales and AAV serotypes tested. These data establish AAVX affinity chromatography as a versatile and efficient method for purification of a broad range of AAV serotypes.
RESUMO
Recombinant adeno-associated viruses (rAAVs) are a preferred vector system in clinical gene transfer. A fundamental challenge to formulate and deliver rAAVs as stable and efficacious vaccines is to elucidate interrelationships between the vector's physicochemical properties and biological potency. To this end, we evaluated an rAAV-based coronavirus disease 2019 (COVID-19) vaccine candidate that encodes the Spike antigen (AC3) and is produced by a commercially viable process. First, state-of-the-art analytical techniques were employed to determine key structural attributes of AC3, including primary and higher-order structures, particle size, empty/full capsid ratios, aggregates, and multi-step thermal degradation pathway analysis. Next, several quantitative potency measures for AC3 were implemented, and data were correlated with the physicochemical analyses on thermally stressed and control samples. Results demonstrate links between decreasing AC3 physical stability profiles, in vitro transduction efficiency in a cell-based assay, and, importantly, in vivo immunogenicity in a mouse model. These findings are discussed in the general context of future development of rAAV-based vaccine candidates as well as specifically for the rAAV vaccine application under study.
RESUMO
Myelinating oligodendrocytes are essential for neuronal communication and homeostasis of the central nervous system (CNS). One of the most abundant molecules in the mammalian CNS is N-acetylaspartate (NAA), which is catabolized into L-aspartate and acetate by the enzyme aspartoacylase (ASPA) in oligodendrocytes. The resulting acetate moiety is thought to contribute to myelin lipid synthesis. In addition, affected NAA metabolism has been implicated in several neurological disorders, including leukodystrophies and demyelinating diseases such as multiple sclerosis. Genetic disruption of ASPA function causes Canavan disease, which is hallmarked by increased NAA levels, myelin and neuronal loss, large vacuole formation in the CNS, and early death in childhood. Although NAA's direct role in the CNS is inconclusive, in peripheral adipose tissue, NAA-derived acetate has been found to modify histones, a mechanism known to be involved in epigenetic regulation of cell differentiation. We hypothesize that a lack of cellular differentiation in the brain contributes to the disruption of myelination and neurodegeneration in diseases with altered NAA metabolism, such as Canavan disease. Our study demonstrates that loss of functional Aspa in mice disrupts myelination and shifts the transcriptional expression of neuronal and oligodendrocyte markers towards less differentiated stages in a spatiotemporal manner. Upon re-expression of ASPA, these oligodendrocyte and neuronal lineage markers are either improved or normalized, suggesting that NAA breakdown by Aspa plays an essential role in the maturation of neurons and oligodendrocytes. Also, this effect of ASPA re-expression is blunted in old mice, potentially due to limited ability of neuronal, rather than oligodendrocyte, recovery.
Assuntos
Doença de Canavan , Camundongos , Animais , Doença de Canavan/genética , Doença de Canavan/metabolismo , Linhagem da Célula , Epigênese Genética , Sistema Nervoso Central/metabolismo , Oligodendroglia , Bainha de Mielina/metabolismo , MamíferosRESUMO
Gene therapy is emerging as a modality in 21st-century medicine. Adeno-associated viral (AAV) gene transfer is a leading technology to achieve efficient and durable expression of a therapeutic transgene. However, the structural complexity of the capsid has constrained efforts to engineer the particle toward improved clinical safety and efficacy. Here, we generate a curated library of barcoded AAVs with mutations across a variety of functionally relevant motifs. We then screen this library in vitro and in vivo in mice and nonhuman primates, enabling a broad, multiparametric assessment of every vector within the library. Among the results, we note a single residue that modulates liver transduction across all interrogated models while preserving transduction in heart and skeletal muscles. Moreover, we find that this mutation can be grafted into AAV9 and leads to profound liver detargeting while retaining muscle transduction-a finding potentially relevant to preventing hepatoxicities seen in clinical studies.
Assuntos
Capsídeo , Vetores Genéticos , Animais , Camundongos , Capsídeo/metabolismo , Vetores Genéticos/genética , Dependovirus/genética , Proteínas do Capsídeo/genética , Fígado/metabolismoRESUMO
Inner ear gene therapy using adeno-associated viral vectors (AAV) promises to alleviate hearing and balance disorders. We previously established the benefits of Anc80L65 in targeting inner and outer hair cells in newborn mice. To accelerate translation to humans, we now report the feasibility and efficiency of the surgical approach and vector delivery in a nonhuman primate model. Five rhesus macaques were injected with AAV1 or Anc80L65 expressing eGFP using a transmastoid posterior tympanotomy approach to access the round window membrane after making a small fenestra in the oval window. The procedure was well tolerated. All but one animal showed cochlear eGFP expression 7-14 days following injection. Anc80L65 in 2 animals transduced up to 90% of apical inner hair cells; AAV1 was markedly less efficient at equal dose. Transduction for both vectors declined from apex to base. These data motivate future translational studies to evaluate gene therapy for human hearing disorders.
Assuntos
Dependovirus , Vetores Genéticos , Animais , Cóclea/fisiologia , Dependovirus/genética , Técnicas de Transferência de Genes , Terapia Genética/métodos , Vetores Genéticos/genética , Macaca mulatta/genética , CamundongosRESUMO
Adeno-associated viral (AAV) vectors hold great potential for liver-directed gene therapy. Stable and high levels of transgene expression have been achieved in many murine models. Systemic delivery of AAV vectors in nonhuman primates (NHPs) that are natural hosts of AAVs appear to be challenging due to the high prevalence of pre-existing neutralizing antibodies (NAbs). This study evaluates the performance of AAV8, hu.37, and rh.8 vectors expressing green fluorescent protein (GFP) from a liver-specific promoter in rhesus macaques. Two of the animals that received AAV8 showed transduction of 24 and 40% of hepatocytes 7 days after systemic vector delivery. Importantly, expression was detected in several animals after 35 days despite the elevation of liver enzymes and development of transgene-specific T cells in liver. Pre-existing low levels of NAbs profoundly impacted the outcome of gene transfer and redirected vector DNA to spleen. We developed a sensitive in vivo passive transfer assay to detect low levels of NAbs to these novel AAV serotypes. Other strategies need to be developed to reduce immune response to the transgene in order to maintain long-term gene expression.
Assuntos
Dependovirus/genética , Dependovirus/imunologia , Fígado/metabolismo , Transdução Genética/métodos , Animais , Anticorpos Neutralizantes/metabolismo , Vetores Genéticos/genética , Vetores Genéticos/imunologia , Proteínas de Fluorescência Verde , MacacaRESUMO
Infants and older adults are especially vulnerable to infection by respiratory syncytial virus (RSV), which can cause significant illness and irreparable damage to the lower respiratory tract and for which an effective vaccine is not readily available. Palivizumab, a recombinant monoclonal antibody (mAb), is an approved therapeutic for RSV infection for use in high-risk infants only. Due to several logistical issues, including cost of goods and scale-up limitations, palivizumab is not approved for other populations that are vulnerable to severe RSV infections, such as older adults. In this study, we demonstrate that intranasal delivery of adeno-associated virus serotype 9 (AAV9) vector expressing palivizumab or motavizumab, a second-generation version of palivizumab, significantly reduced the viral load in the lungs of the BALB/c mouse model of RSV infection. Notably, we demonstrate that AAV9 vector-mediated prophylaxis against RSV was effective despite the presence of serum-circulating neutralizing AAV9 antibodies. These findings substantiate the feasibility of repeatedly administering AAV9 vector to the airway for seasonal prophylaxis against RSV, thereby expanding the application of vectored delivery of mAbs as an effective prophylaxis strategy against various airborne viruses.
Assuntos
Dependovirus , Infecções por Vírus Respiratório Sincicial , Animais , Antivirais , Dependovirus/genética , Pulmão , Camundongos , Camundongos Endogâmicos BALB C , Palivizumab/uso terapêutico , Infecções por Vírus Respiratório Sincicial/tratamento farmacológico , Infecções por Vírus Respiratório Sincicial/prevenção & controleRESUMO
The SARS-CoV-2 pandemic has affected more than 185 million people worldwide resulting in over 4 million deaths. To contain the pandemic, there is a continued need for safe vaccines that provide durable protection at low and scalable doses and can be deployed easily. Here, AAVCOVID-1, an adeno-associated viral (AAV), spike-gene-based vaccine candidate demonstrates potent immunogenicity in mouse and non-human primates following a single injection and confers complete protection from SARS-CoV-2 challenge in macaques. Peak neutralizing antibody titers are sustained at 1 year and complemented by functional memory T cell responses. The AAVCOVID vector has no relevant pre-existing immunity in humans and does not elicit cross-reactivity to common AAVs used in gene therapy. Vector genome persistence and expression wanes following injection. The single low-dose requirement, high-yield manufacturability, and 1-month stability for storage at room temperature may make this technology well suited to support effective immunization campaigns for emerging pathogens on a global scale.
Assuntos
Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Vacinas contra COVID-19/imunologia , COVID-19/prevenção & controle , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Vacinas contra COVID-19/administração & dosagem , Vacinas contra COVID-19/genética , Dependovirus/genética , Dependovirus/metabolismo , Feminino , Humanos , Imunogenicidade da Vacina/imunologia , Memória Imunológica/imunologia , Macaca fascicularis , Macaca mulatta , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Linfócitos T/imunologia , Transgenes/genética , Vacinação/métodos , Carga Viral/imunologiaRESUMO
The SARS-CoV-2 pandemic has affected more than 70 million people worldwide and resulted in over 1.5 million deaths. A broad deployment of effective immunization campaigns to achieve population immunity at global scale will depend on the biological and logistical attributes of the vaccine. Here, two adeno-associated viral (AAV)-based vaccine candidates demonstrate potent immunogenicity in mouse and nonhuman primates following a single injection. Peak neutralizing antibody titers remain sustained at 5 months and are complemented by functional memory T-cells responses. The AAVrh32.33 capsid of the AAVCOVID vaccine is an engineered AAV to which no relevant pre-existing immunity exists in humans. Moreover, the vaccine is stable at room temperature for at least one month and is produced at high yields using established commercial manufacturing processes in the gene therapy industry. Thus, this methodology holds as a very promising single dose, thermostable vaccine platform well-suited to address emerging pathogens on a global scale.
RESUMO
Achieving efficient cardiac gene transfer in a large animal model has proven to be technically challenging. Previous strategies have used cardiopulmonary bypass or dual catheterization with the aid of vasodilators to deliver vectors, such as adenovirus, adeno-associated virus (AAV), or plasmid DNA. Although single-stranded AAV (ssAAV) vectors have shown the greatest promise, they suffer from delayed expression, which might be circumvented using self-complementary vectors. We sought to optimize cardiac gene transfer using a percutaneous transendocardial injection catheter to deliver adeno-associated viral vectors to the canine myocardium. Four vectors were evaluated--ssAAV9, self-complementary AAV9 (scAAV9), scAAV8, scAAV6--so that comparison could be made between single-stranded and self-complementary vectors as well as among serotypes 9, 8, and 6. We demonstrate that scAAV is superior to ssAAV and that AAV 6 is superior to the other serotypes evaluated. Biodistribution studies revealed that vector genome copies were 15-4,000 times more abundant in the heart than in any other organ for scAAV6. Percutaneous transendocardial injection of scAAV6 is a safe, effective method to achieve efficient cardiac gene transfer.
Assuntos
Dependovirus/genética , Miocárdio/metabolismo , Transgenes/genética , Animais , Cães , Endotélio/metabolismo , Vetores Genéticos/genética , CoraçãoRESUMO
Achieving efficient cardiac gene transfer in a large animal model has proven to be technically challenging. Previous strategies have used cardiopulmonary bypass or dual catheterization with the aid of vasodilators to deliver vectors, such as adenovirus, adeno-associated virus (AAV), or plasmid DNA. Although single-stranded AAV (ssAAV) vectors have shown the greatest promise, they suffer from delayed expression, which might be circumvented using self-complementary vectors. We sought to optimize cardiac gene transfer using a percutaneous transendocardial injection catheter to deliver adeno-associated viral vectors to the canine myocardium. Four vectors were evaluated-ssAAV9, self-complementary AAV9 (scAAV9), scAAV8, scAAV6-so that comparison could be made between single-stranded and self-complementary vectors as well as among serotypes 9, 8, and 6. We demonstrate that scAAV is superior to ssAAV and that AAV 6 is superior to the other serotypes evaluated. Biodistribution studies revealed that vector genome copies were 15-4,000 times more abundant in the heart than in any other organ for scAAV6. Percutaneous transendocardial injection of scAAV6 is a safe, effective method to achieve efficient cardiac gene transfer.
RESUMO
The adeno-associated viral vector (AAV) platform has developed into a primary modality for efficient in vivo, and in more limited settings, in vitro or ex vivo gene transfer. Its applications range from a tool for experimental purposes to preclinical and clinical gene therapy. The ability to accurately and reproducibly quantify vector concentration is critical for any of these applications. While several quantification assays are available, here we outline a detailed protocol for the quantification of DNase-I protected vector genomes reliant on the polymerase chain reaction (PCR) as a measure of the active component of the vector, namely its transgene cargo. With the emergence of droplet digital PCR (ddPCR), we provide side-by-side protocols for traditional TaqMan™ real-time, quantitative PCR (qPCR) and ddPCR, as well as comparative data generated with both methods. Lastly, we discuss the importance of the use of surfactant (here, Pluronic® F-68) in the execution of the assay to limit DNA and AAV adherence to various carriers during the titration, particularly at low concentrations. We believe these protocols can lead to reduced variability and increased comparability between AAV studies.
Assuntos
Dependovirus/genética , Vetores Genéticos/genética , Genoma Viral , Reação em Cadeia da Polimerase em Tempo Real , Carga Viral , Humanos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase em Tempo Real/normas , Sensibilidade e Especificidade , Fluxo de TrabalhoRESUMO
BACKGROUND: The atheroprotective effects of systemic delivery of either apolipoprotein A-I (wtApoA-I) or the naturally occurring mutant ApoA-I Milano (ApoA-IM) have been established in animal and human trials, but direct comparison studies evaluating the phenotype of ApoA-I or ApoAI-Milano knock-in mice or bone marrow transplantated animals with selectively ApoA-I or ApoAI-Milano transduced macrophages give conflicting results regarding the superior performance of either one. We therefore sought to compare the two forms of apoA-I using liver-directed somatic gene transfer in hypercholesterinemic mice--a model which is most adequately mimicking the clinical setting. METHODS AND RESULTS: Vectors based on AAV serotype 8 (AAV2.8) encoding wtApoA-I, ApoA-IM or green fluorescent protein (GFP) as control were constructed. LDL receptor deficient mice were fed a Western diet. After 8 weeks the AAV vectors were injected, and 6 weeks later atherosclerotic lesion size was determined by aortic en face analysis. Expression of wtApoA-I reduced progression of atherosclerosis by 32% compared with control (p = 0.02) and of ApoA-IM by 24% (p = 0.04). There was no significant difference between the two forms of ApoA-I in inhibiting atherosclerosis progression. CONCLUSION: Liver-directed AAV2.8-mediated gene transfer of wtApoA-I and ApoA-IM each significantly reduced atherosclerosis progression to a similar extent.
Assuntos
Apolipoproteína A-I/genética , Aterosclerose/genética , Aterosclerose/metabolismo , Transdução Genética , Animais , Aterosclerose/prevenção & controle , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Humanos , Hipercolesterolemia/genética , Hipercolesterolemia/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Mutação , Receptores de LDL/deficiência , Receptores de LDL/genética , Transdução Genética/métodosRESUMO
Interleukin-36 (IL-36) represents three cytokines, IL-36α, IL-36ß and IL-36γ, which bind to the same receptor, IL-1RL2; however, their physiological function(s) remain poorly understood. Here, the role of IL-36 in immunity against HSV-1 was examined using the flank skin infection mouse model. Expression analyses revealed increased levels of IL-36α and IL-36ß mRNA in infected skin, while constitutive IL-36γ levels remained largely unchanged. In human keratinocytes, IL-36α mRNA was induced by HSV-1, while IL-1ß and TNFα increased all three IL-36 mRNAs. The dominant alternative splice variant of human IL-36ß mRNA was isoform 2, which is the ortholog of the known mouse IL-36ß mRNA. Mice deficient in IL-36ß, but not IL-36α or IL-36γ, succumbed more frequently to HSV-1 infection than wild type mice. Furthermore, IL-36ß-/- mice developed larger zosteriform skin lesions along infected neurons. Levels of HSV-1 specific antibodies, CD8+ cells and IFNγ-producing CD4+ cells were statistically equal in wild type and IL-36ß-/- mice, suggesting similar initiation of adaptive immunity in the two strains. This correlated with the time at which HSV-1 genome and mRNA levels in primary skin lesions started to decline in both wild type and IL-36ß-/- mice. Our data indicate that IL-36ß has previously unrecognized functions protective against HSV-1 infection.
Assuntos
Herpes Simples/imunologia , Herpes Simples/patologia , Herpesvirus Humano 1/imunologia , Imunidade Inata , Interleucina-1/metabolismo , Imunidade Adaptativa , Animais , Modelos Animais de Doenças , Interleucina-1/deficiência , Camundongos , Camundongos Knockout , Pele/patologia , Análise de SobrevidaRESUMO
Canavan disease (CD) is a debilitating and lethal leukodystrophy caused by mutations in the aspartoacylase (ASPA) gene and the resulting defect in N-acetylaspartate (NAA) metabolism in the CNS and peripheral tissues. Recombinant adeno-associated virus (rAAV) has the ability to cross the blood-brain barrier and widely transduce the CNS. We developed a rAAV-based and optimized gene replacement therapy, which achieves early, complete, and sustained rescue of the lethal disease phenotype in CD mice. Our treatment results in a super-mouse phenotype, increasing motor performance of treated CD mice beyond that of WT control mice. We demonstrate that this rescue is oligodendrocyte independent, and that gene correction in astrocytes is sufficient, suggesting that the establishment of an astrocyte-based alternative metabolic sink for NAA is a key mechanism for efficacious disease rescue and the super-mouse phenotype. Importantly, the use of clinically translatable high-field imaging tools enables the noninvasive monitoring and prediction of therapeutic outcomes for CD and might enable further investigation of NAA-related cognitive function.
Assuntos
Amidoidrolases/genética , Doença de Canavan/terapia , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Amidoidrolases/metabolismo , Animais , Ácido Aspártico/análogos & derivados , Barreira Hematoencefálica/metabolismo , Doença de Canavan/genética , Doença de Canavan/metabolismo , Sistema Nervoso Central , Dependovirus/genética , Modelos Animais de Doenças , Humanos , Camundongos , Oligodendroglia/metabolismoRESUMO
Herpes simplex virus-1 (HSV-1) is a human pathogen that utilizes several strategies to circumvent the host immune response. An immune evasion mechanism employed by HSV-1 is retention of interleukin-1ß (IL-1ß) in the intracellular space, which blocks the pro-inflammatory activity of IL-1ß. Here we report that HSV-1-infected keratinocytes actively release the also pro-inflammatory IL-1α, preserving the ability of infected cells to signal danger to the surrounding tissue. The extracellular release of IL-1α is independent of inflammatory caspases. In vivo recruitment of leukocytes to early HSV-1 microinfection sites within the epidermis is dependent upon IL-1 signalling. Following cutaneous HSV-1 infection, mice unable to signal via extracellular IL-1α exhibit an increased mortality rate associated with viral dissemination. We conclude that IL-1α acts as an alarmin essential for leukocyte recruitment and protective immunity against HSV-1. This function may have evolved to counteract an immune evasion mechanism deployed by HSV-1.