RESUMEN
Sepsis-associated acute kidney injury (S-AKI) is a frequent complication in patients who are critically ill, which is often initiated by glomerular endothelial cell dysfunction. Although transient receptor vanilloid subtype 4 (TRPV4) ion channels are known to be permeable to Ca2+ and are widely expressed in the kidneys, the role of TRPV4 on glomerular endothelial inflammation in sepsis remains elusive. In the present study, we found that TRPV4 expression in mouse glomerular endothelial cells (MGECs) increased after lipopolysaccharide (LPS) stimulation or cecal ligation and puncture challenge, which increased intracellular Ca2+ in MGECs. Furthermore, the inhibition or knockdown of TRPV4 suppressed LPS-induced phosphorylation and translocation of inflammatory transcription factors NF-κB and IRF-3 in MGECs. Clamping intracellular Ca2+ mimicked LPS-induced responses observed in the absence of TRPV4. In vivo experiments showed that the pharmacologic blockade or knockdown of TRPV4 reduced glomerular endothelial inflammatory responses, increased survival rate, and improved renal function in cecal ligation and puncture-induced sepsis without altering renal cortical blood perfusion. Taken together, our results suggest that TRPV4 promotes glomerular endothelial inflammation in S-AKI and that its inhibition or knockdown alleviates glomerular endothelial inflammation by reducing Ca2+ overload and NF-κB/IRF-3 activation. These findings provide insights that may aid in the development of novel pharmacologic strategies for the treatment of S-AKI.
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Lesión Renal Aguda , Antineoplásicos , Sepsis , Ratones , Animales , FN-kappa B/metabolismo , Células Endoteliales/metabolismo , Lipopolisacáridos/farmacología , Canales Catiónicos TRPV/metabolismo , Lesión Renal Aguda/etiología , Lesión Renal Aguda/metabolismo , Inflamación/metabolismo , Sepsis/complicaciones , Sepsis/metabolismoRESUMEN
BACKGROUND: Exercise training protects the heart against pathological cardiac remodeling and confers cardioprotection from heart failure. However, the underlying mechanism is still elusive. METHODS: An integrative analysis of multi-omics data of the skeletal muscle in response to exercise is performed to search for potential exerkine. Then, CCDC80tide is examined in humans after acute exercise. The role of CCDC80tide is assessed in a mouse model of hypertensive cardiac remodeling and in hypertension-mediated cell injury models. The transcriptomic analysis and immunoprecipitation assay are conducted to explore the mechanism. FINDINGS: The coiled-coil domain-containing protein 80 (CCDC80) is found strongly positively associated with exercise. Interestingly, exercise stimuli induce the secretion of C-terminal CCDC80 (referred as CCDC80tide hereafter) via EVs-encapsulated CCDC80tide into the circulation. Importantly, cardiac-specific expression of CCDC80tide protects against angiotensin II (Ang II)-induced cardiac hypertrophy and fibrosis in mice. In in vitro studies, the expression of CCDC80tide reduces Ang II-induced cardiomyocyte hypertrophy, cardiac microvascular endothelial cell (CMEC) inflammation, and mitigated vascular smooth muscle cell (VSMC) proliferation and collagen formation. To understand the cardioprotective effect of CCDC80tide, a transcriptomic analysis reveals a dramatic inhibition of the STAT3 (Signal transducer and activator of transcription 3) signaling pathway in CCDC80tide overexpressing cells. Mechanistically, CCDC80tide selectively interacts with the kinase-active form of JAK2 (Janus kinase 2) and consequently inhibits its kinase activity to phosphorylate and activate STAT3. INTERPRETATION: The results provide new insights into exercise-afforded cardioprotection in pathological cardiac remodeling and highlight the therapeutic potential of CCDC80tide in heart failure treatment. FUNDING: This work was supported by the National Natural Science Foundation of China [Grant/Award Numbers: 81770428, 81830010, 82130012, 81900438, 82100447); Shanghai Science and Technology Committee [Grant/Award Numbers: 21S11903000, 19JC1415702]; Emerging and Advanced Technology Programs of Hospital Development Center of Shanghai [Grant/Award Number: SHDC12018129]; China Postdoctoral Science Foundation [2021M692108]; and China National Postdoctoral Program for Innovative Talents [BX20200211].
Asunto(s)
Insuficiencia Cardíaca , Hipertensión , Angiotensina II/farmacología , Animales , China , Proteínas de la Matriz Extracelular/metabolismo , Corazón , Insuficiencia Cardíaca/metabolismo , Humanos , Hipertensión/metabolismo , Ratones , Miocitos Cardíacos/metabolismo , Remodelación VentricularRESUMEN
Purpose: Activation of NLR (nucleotide-binding and leucine-rich repeat immune receptor) family pyrin domain containing 3 (NLRP3) inflammasome mediating interleukin- (IL-) 1ß secretion has emerged as an important component of inflammatory processes in atherogenesis. The nuclear receptor Nur77 is highly expressed in human atherosclerotic lesions; however, its functional role in macrophage NLRP3 inflammasome activation has not yet been clarified. Methods, Materials, and Results. Eight-week-old apolipoprotein E (ApoE)-/- and ApoE-/- Nur77-/- mice that were fed a Western diet underwent partial ligation of the left common carotid artery (LCCA) and left renal artery (LRA) to induce atherogenesis. Four weeks later, severe plaque burden associated with increased lipid deposition, reduced smooth muscle cells, macrophage infiltration, and decreased collagen expression was identified in ApoE-/- Nur77-/- mice compared with those in ApoE-/- mice. ApoE-/- Nur77-/- mice showed increased macrophage inflammatory responses in carotid atherosclerotic lesions. In vitro studies demonstrated that oxidized low-density lipoprotein cholesterol (ox-LDL) increased the release of lactate dehydrogenase (LDH) and upregulated the expressions of cleaved caspase-1, cleaved IL-1ß and gasdermin D (GSMD) in WT peritoneal macrophages (PMs) in a NLRP3-dependent manner. Nur77-/- PMs exhibited a further increased level of NLRP3 inflammasome-mediated inflammation under ox-LDL treatment compared with WT PMs. Mechanistically, Nur77 could bind to the promoter of NLRP3 and inhibit its transcriptional activity. Conclusions: This study demonstrated that Nur77 deletion promotes atherogenesis by exacerbating NLRP3 inflammasome-mediated inflammation.
Asunto(s)
Aterosclerosis , Inflamasomas , Animales , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Aterosclerosis/patología , Colesterol/metabolismo , Inflamasomas/metabolismo , Inflamación/patología , Interleucina-1beta/metabolismo , Macrófagos/metabolismo , Ratones , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores NuclearesRESUMEN
PURPOSE: Macrophage apoptosis coupled with a defective phagocytic clearance of the apoptotic cells promotes plaque necrosis in advanced atherosclerosis, which causes acute atherothrombotic vascular disease. Nonsteroidal anti-inflammatory drug sulindac derivative K-80003 treatment was previously reported to dramatically attenuate atherosclerotic plaque progression and destabilization. However, the underlying mechanisms are not fully understood. This study aimed to determine the role of K-80003 on macrophage apoptosis and elucidate the underlying mechanism. METHODS: The mouse model of vulnerable carotid plaque in ApoE-/- mice was developed in vivo. Consequently, mice were randomly grouped into two study groups: the control group and the K-80003 group (30 mg/kg/day). Samples of carotid arteries were collected to determine atherosclerotic necrotic core area, cellular apoptosis, and oxidative stress. The effects of K-80003 on RAW264.7 macrophage apoptosis, oxidative stress, and autophagic flux were also examined in vitro. RESULTS: K-80003 significantly suppressed necrotic core formation and inhibited cellular apoptosis of vulnerable plaques. K-80003 can also inhibit 7-ketocholesterol-induced macrophage apoptosis in vitro. Furthermore, K-80003 inhibited intraplaque cellular apoptosis mainly through the suppression of oxidative stress, which is a key cause of advanced lesional macrophage apoptosis. Mechanistically, K-80003 prevented 7-ketocholesterol-induced impairment of autophagic flux in macrophages, evidenced by the decreased LC3II and SQSTM1/p62 expression, GFP-RFP-LC3 cancellation upon K-80003 treatment. CONCLUSION: Inhibition of macrophage apoptosis and necrotic core formation by autophagy-mediated reduction of oxidative stress is one mechanism of the suppression of plaque progression and destabilization by K-80003.
Asunto(s)
Aterosclerosis , Placa Aterosclerótica , Animales , Ratones , Apoptosis , Aterosclerosis/tratamiento farmacológico , Aterosclerosis/metabolismo , Macrófagos/metabolismo , Ratones Endogámicos C57BL , Necrosis/metabolismo , Placa Aterosclerótica/metabolismo , Sulindac/metabolismo , Sulindac/farmacologíaRESUMEN
AIMS: Pathological cardiac hypertrophy induced by activation of the renin-angiotensin-aldosterone system (RAAS) is one of the leading causes of heart failure. However, in current clinical practice, the strategy for targeting the RAAS is not sufficient to reverse hypertrophy. Here, we investigated the effect of prostaglandin E1 (PGE1) on angiotensin II (AngII)-induced cardiac hypertrophy and potential molecular mechanisms underlying the effect. METHODS AND RESULTS: Adult male C57 mice were continuously infused with AngII or saline and treated daily with PGE1 or vehicle for two weeks. Neonatal rat cardiomyocytes were cultured to detect AngII-induced hypertrophic responses. We found that PGE1 ameliorated AngII-induced cardiac hypertrophy both in vivo and in vitro. The RNA sequencing (RNA-seq) and expression pattern analysis results suggest that Netrin-1 (Ntn1) is the specific target gene of PGE1. The protective effect of PGE1 was eliminated after knockdown of Ntn1. Moreover, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that the PGE1-mediated signaling pathway changes are associated with the mitogen-activated protein kinase (MAPK) pathway. PGE1 suppressed AngII-induced activation of the MAPK signaling pathway, and such an effect was attenuated by Ntn1 knockdown. Blockade of MAPK signaling rescued the phenotype of cardiomyocytes caused by Ntn1 knockdown, indicating that MAPK signaling may act as the downstream effector of Ntn1. Furthermore, inhibition of the E-prostanoid (EP) 3 receptor, as opposed to the EP1, EP2, or EP4 receptor, in cardiomyocytes reversed the effect of PGE1, and activation of EP3 by sulprostone, a specific agonist, mimicked the effect of PGE1. CONCLUSION: In conclusion, PGE1 ameliorates AngII-induced cardiac hypertrophy through activation of the EP3 receptor and upregulation of Ntn1, which inhibits the downstream MAPK signaling pathway. Thus, targeting EP3, as well as the Ntn1-MAPK axis, may represent a novel approach for treating pathological cardiac hypertrophy.
Asunto(s)
Alprostadil/farmacología , Angiotensina II/farmacología , Cardiomegalia/inducido químicamente , Cardiomegalia/genética , Netrina-1/genética , Subtipo EP3 de Receptores de Prostaglandina E/genética , Regulación hacia Arriba/efectos de los fármacos , Animales , Masculino , Ratones , Ratones Endogámicos C57BL , Miocitos Cardíacos/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacos , Regulación hacia Arriba/genéticaRESUMEN
BACKGROUND: Oxidative stress plays critical pathophysiological roles in vascular remodeling-related cardiovascular diseases, including hypertension, atherosclerosis, and restenosis. Previous studies demonstrate that SENP3, a redox-sensitive SUMO2/3-specific protease, is strongly implicated in cancer development and progression. However, the role of SENP3 in vascular remodeling remains unknown. METHODS: We generated three mouse models of vascular remodeling due to low shear stress, hypertension, and atherosclerosis. The expression of SENP3 was determined by western blotting and/or immunofluorescence staining in cultured vascular smooth muscle cells (VSMCs), animal models, and human samples. The biological function of SENP3 in proliferation and migration of VSMC and vascular remodeling was further investigated in vitro and in vivo models. FINDINGS: SENP3 was highly expressed in VSMCs of remodeled arteries, accompanied by elevated reactive oxygen species (ROS) levels. In cultured VSMCs, SENP3 protein levels were enhanced by oxidized low-density lipoprotein and Angiotensin II in a ROS-dependent manner. SENP3 overexpression significantly promoted and sh-RNA-mediated knockdown markedly inhibited VSMCs proliferation and migration. Immunofluorescence staining showed that SENP3 expression was correlated with intimal area in remodeled arteries. Furthermore, we demonstrated that SENP3 interacted with ß-catenin and inhibited its proteasome-dependent degradation via de-SUMOylation of ß-catenin. Most importantly, SENP3+/- mice exhibited alleviated vascular remodeling. INTERPRETATION: Our results highlight the important function of SENP3 as a redox sensor and mediator in vascular remodeling.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Remodelación Vascular , Animales , Proliferación Celular , Células Cultivadas , Cisteína Endopeptidasas/genética , Células HEK293 , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , Estrés Oxidativo , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismo , Sumoilación , beta Catenina/metabolismoRESUMEN
PURPOSE: Autophagy inhibition is a novel therapeutic strategy suggested for patients with advanced cancer, especially those who have undergone radiation therapy. In the present study, we investigated whether autophagy inhibitors accelerate the progression of radiation-associated atherosclerosis (RAA). METHODS AND MATERIALS: Eight-week-old apolipoprotein (ApoE-/-) mice were fed a Western diet, and their left common carotid arteries were partially ligated to induce atherogenesis. Four weeks later, local ionizing radiation (IR) at a dose of 5 or 10 Gy was used to induce RAA in the left common carotid artery. After another 4 weeks, severe plaque burden associated with increased macrophage infiltration and lipid deposition, reduced smooth muscle cells, and decreased collagen expression was observed. In addition, these changes occurred in a dose-dependent manner. Improved autophagic flux caused by IR was observed in both macrophages of the atherosclerotic plaque and peritoneal macrophages in vitro. The inhibition of autophagic flux by chloroquine (50 mg/kg/d) further accelerated the progression of RAA in the left common carotid arteries of ApoE-/- mice. Furthermore, chloroquine treatment exacerbated IR-induced p65 nuclear translocation, IκBα degradation, and transcription of nuclear factor-κB (NF-κB) target genes in peritoneal macrophages. CONCLUSIONS: IR promotes atherogenesis and increases autophagic flux. In addition, autophagy inhibition by chloroquine accelerates the progression of RAA lesions by stimulating NF-κB-mediated inflammatory responses in macrophages.
Asunto(s)
Antineoplásicos/efectos adversos , Aterosclerosis/patología , Autofagia/efectos de los fármacos , Traumatismos por Radiación/patología , Transporte Activo de Núcleo Celular/efectos de los fármacos , Animales , Aterosclerosis/etiología , Aterosclerosis/metabolismo , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Cloroquina/efectos adversos , Progresión de la Enfermedad , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/metabolismo , Ratones , Inhibidor NF-kappaB alfa/metabolismo , Proteolisis/efectos de los fármacos , Traumatismos por Radiación/etiología , Traumatismos por Radiación/metabolismo , Factor de Transcripción ReIA/metabolismoRESUMEN
BACKGROUND AND PURPOSE: Atherosclerosis is a chronic inflammatory disease, and retinoid X receptor-α (RXRα) is an intriguing anti-atherosclerosis target. This study investigated whether and how an RXRα modulator, K-80003, derived from a non-steroidal anti-inflammatory drug attenuates atherosclerotic plaque progression and destabilization. EXPERIMENTAL APPROACH: Our previously established ApoE-/- mouse model of carotid vulnerable plaque progression was treated with K-80003 or vehicle for 4 or 8 weeks. Samples of carotid arteries and serum were collected to determine atherosclerotic lesion size, histological features, expression of related proteins, and lipid profiles. In vitro studies were carried out in 7-ketocholesterol (7-KC)-stimulated macrophages treated with or without K-80003. KEY RESULTS: K-80003 significantly reduced lesion size, plaque rupture, macrophage infiltration, and inflammatory cytokine levels. Plaque macrophages positive for nuclear p65 (RelA) NF-κB subunit were markedly reduced after K-80003 treatment. Also, K-80003 treatment inhibited 7-KC-induced p65 nuclear translocation, IκBα degradation, and transcription of NF-κB target genes. In addition, K-80003 inhibited NF-κB pathway mainly through the reduction of p62/sequestosome 1 (SQSTM1), probably due to promotion of autophagic flux by K-80003. Mechanistically, cytoplasmic localization of RXRα was associated with decreased autophagic flux. Increasing cytoplasmic RXRα expression by overexpression of RXRα/385 mutant decreased autophagic flux in RAW264.7 cells. Finally, K-80003 strongly inhibited 7-KC-induced RXRα cytoplasmic translocation. CONCLUSIONS AND IMPLICATIONS: K-80003 suppressed atherosclerotic plaque progression and destabilization by promoting macrophage autophagic flux and consequently inhibited the p62/SQSTM1-mediated NF-κB proinflammatory pathway. Thus, targeting RXRα-mediated autophagy-inflammation axis by its noncanonical modulator may represent a promising strategy to treat atherosclerosis.
Asunto(s)
Apolipoproteínas E/metabolismo , Placa Aterosclerótica/tratamiento farmacológico , Sulindac/análogos & derivados , Animales , Apolipoproteínas E/deficiencia , Células Cultivadas , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Placa Aterosclerótica/metabolismo , Placa Aterosclerótica/patología , Células RAW 264.7 , Sulindac/administración & dosificación , Sulindac/efectos adversos , Sulindac/farmacologíaRESUMEN
Xuezhikang (XZK), an extract of red yeast rice, is a traditional Chinese medicine widely used for the treatment of cardiovascular diseases in China and other countries. However, whether XZK treatment can improve atherosclerotic plaque stability is not fully understood. Based on our previously developed mouse model of spontaneous vulnerable plaque formation and rupture in carotid arteries in ApoE-/- mice. We showed that low-dose (600 mg/kg/d) XZK improved plaque stability without decreasing plaque area, whereas high-dose (1200 mg/kg/d) XZK dramatically inhibited vulnerable plaque progression accompanied by decreased plaque area. Mechanistically, XZK significantly suppressed lesional endoplasmic reticulum (ER) stress in mouse carotid arteries. In vitro, XZK inhibited 7-KC-induced activation of ER stress in RAW264.7 macrophages, as assessed by the reduced levels of p-PERK, p-IRE1α, p-eIF2α, c-ATF6, s-XBP1, and CHOP. Compared to controls, the XZK-treated group displayed dramatically decreased apoptotic cell numbers (shown by decreased TUNEL- and cleaved caspase3-positive cells), lower necrotic core area and ratio, and reduced expression of NF-κB target gene. In RAW264.7 cells, XZK inhibited 7-KC-induced upregulation of apoptosis, protein expression of apoptotic markers (cleaved caspase-3 and cleaved PARP), and NF-κB activation (shown by target gene transcription and IκBα reduction). Collectively, our results suggest that XZK effectively suppresses vulnerable plaque progression and rupture by mitigating macrophage ER stress and consequently inhibiting apoptosis and the NF-κB pro-inflammatory pathway, thereby providing an alternative therapeutic strategy for stabilizing atherosclerotic plaques.
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Apoptosis/efectos de los fármacos , Productos Biológicos/química , Medicamentos Herbarios Chinos/farmacología , Estrés del Retículo Endoplásmico/efectos de los fármacos , Inflamación/prevención & control , Placa Aterosclerótica/prevención & control , Animales , Apolipoproteínas E/genética , Progresión de la Enfermedad , Ratones , Ratones Noqueados , FN-kappa B/metabolismo , Células RAW 264.7 , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacosRESUMEN
An anti-rabies virus single-chain antibody fragment of an anti-glycoprotein with the VL-linker-VH orientation, designated scFv57RN, was successfully and conveniently prepared in this study. The scFv57RN protein was mainly expressed in inclusion bodies in Escherichia coli. After washing and purification, the inclusion bodies were finally obtained with an on-column refolding procedure. Further purification by gel exclusion chromatography was performed to remove inactive multimers. About 360 mg of final product was recovered from 1 L of bacterial culture. The final product showed a high neutralizing titer of 950 IU/mg to the CVS-11 strain as measured using the rapid fluorescent focus inhibition test. Our study demonstrated a highly efficient method to mass produce scFV57RN with activity from inclusion bodies, which may be applied in the purification of other insoluble proteins.
Asunto(s)
Expresión Génica , Glicoproteínas/análisis , Replegamiento Proteico , Virus de la Rabia/química , Anticuerpos de Cadena Única , Proteínas Virales/antagonistas & inhibidores , Escherichia coli , Glicoproteínas/química , Cuerpos de Inclusión/química , Cuerpos de Inclusión/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Anticuerpos de Cadena Única/biosíntesis , Anticuerpos de Cadena Única/química , Anticuerpos de Cadena Única/aislamiento & purificación , Proteínas Virales/químicaRESUMEN
Lethal rabies can be prevented effectively by post-exposure prophylactic (PEP) with rabies immunoglobulin (RIG). Single-chain variable fragment (scFv), which is composed of a variable heavy chain (VH) and variable light chain (VL) connected by a peptide linker, may be developed as alternative to RIG for neutralizing rabies virus (RV). However, our previously constructed scFv (FV57S) with the (NH2) VH-linker-VL (COOH) orientation showed a lower neutralizing potency than its parent RIG. This orientation may inhibit FV57S from refolding into an intact and correct conformation. Therefore, the RFV57S protein with a VL-linker-VH orientation was constructed based on FV57S. A HIS tag was incorporated to aid in purification and detection of RFV57S and FV57S. However, abilities of RFV57S and FV57S to bind with the anti-HIS tag mAb were different. Therefore, a novel direct ELISA was established by utilizing a biotin-labeled truncated glycoprotein of RV. Although with similar stability and in vitro neutralizing potency as FV57S, RFV57S showed enhanced binding ability, affinity and in vivo protective efficacy against lethal dose of RV. Our studies support the feasibility of developing a scFv with reversed orientation and provide a novel method for evaluating the binding ability, stability and affinity of engineered antibodies recognizing linear epitope.
Asunto(s)
Glicoproteínas/inmunología , Virus de la Rabia/metabolismo , Rabia/prevención & control , Anticuerpos de Cadena Única/genética , Animales , Anticuerpos Neutralizantes/genética , Anticuerpos Neutralizantes/metabolismo , Anticuerpos Antivirales/genética , Anticuerpos Antivirales/metabolismo , Ratones , Péptidos/genética , Péptidos/metabolismo , Virus de la Rabia/genética , Virus de la Rabia/inmunología , Anticuerpos de Cadena Única/metabolismo , Proteínas Virales/inmunología , Vacunas Virales/administración & dosificaciónRESUMEN
Fatal rabies can be prevented effectively by post-exposure prophylactic (PEP) with rabies immunoglobulin (RIG). Single-chain variable fragments (scFv), which are composed of a variable heavy chain (VH) and a variable light chain (VL) connected by a peptide linker, can potentially be used to replace RIG. However, in our previous study, a scFv (scFV57S) specific for the rabies virus (RV) G protein showed a lower neutralizing potency than that of its parent IgG due to lower stability and altered peptide assembly pattern. In monoclonal antibodies, the VH and VL interact non-covalently, while in scFvs the VH is connected covalently with the VL by the artificial linker. In this study, we constructed and expressed two peptides 57VL-JUN-HIS and 57VH-FOS-HA in Escherichia coli. The well-known Fos and Jun leucine zippers were utilized to dimerize VH and VL similarly to the IgG counterpart. The two peptides assembled to form zipFv57S in vitro. Due to the greater similarity in structure with IgG, the zipFv57S protein showed a higher binding ability and affinity resulting in notable improvement of in vitro neutralizing activity over its corresponding scFv. The zipFv57S protein was also found to be more stable and showed similar protective rate as RIG in mice challenged with a lethal dose of RV. Our results not only indicated zipFv57S as an ideal alternative for RIG in PEP but also offered a novel and efficient hetero-dimerization pattern of VH and VL leading to enhanced neutralizing potency.
Asunto(s)
Anticuerpos Neutralizantes/inmunología , Leucina Zippers/genética , Vacunas Antirrábicas/inmunología , Rabia/prevención & control , Anticuerpos de Cadena Única/inmunología , Proteínas del Envoltorio Viral/antagonistas & inhibidores , Animales , Anticuerpos Neutralizantes/administración & dosificación , Anticuerpos Neutralizantes/genética , Cricetulus , Escherichia coli/genética , Escherichia coli/metabolismo , Femenino , Expresión Génica , Células HEK293 , Humanos , Inmunización , Inmunoglobulina G/administración & dosificación , Leucina Zippers/inmunología , Ratones , Pruebas de Neutralización , Plásmidos/química , Plásmidos/metabolismo , Multimerización de Proteína , Rabia/inmunología , Rabia/virología , Vacunas Antirrábicas/administración & dosificación , Vacunas Antirrábicas/genética , Virus de la Rabia/inmunología , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Anticuerpos de Cadena Única/administración & dosificación , Anticuerpos de Cadena Única/genética , Análisis de Supervivencia , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/inmunologíaRESUMEN
Rabies virus (RV) causes a fatal infectious disease requiring efficient post-exposure prophylaxis (PEP), which includes a rabies vaccine and rabies immunoglobulin (RIG). The single-chain antibody variable fragment (scFv), a small engineered antibody fragment derived from an antibody variable heavy chain and light chain, has the potential to replace the current application of RIG. In previous studies, we constructed and evaluated an anti-rabies virus G protein scFv (FV57) based on the monoclonal antibody CR57. Of the five cysteines in FV57, four are linked in intra-chain disulfide bonds (Cys-VH28/Cys-VH98 and Cys-VL16/Cys-VL84), and one is free (Cys-VL85). However, the thiol in Cys-VL85 neighboring Cys-VL84 in the CDR3 of the light chain is likely to mismatch with the thiol in Cys-VL16 during the renaturing process. In order to study effects of the mismatched disulfide bond, Cys-VL85 and Cys-VL84 of FV57 were mutated to serine to construct mutants FV57(VL85S) and FV57(VL84S). Furthermore, the disulfide bonds in the light chain of FV57, FV57(VL85S) and FV57(VL84S) were deleted by mutating Cys-VL16 to serine. All mutants were prepared and evaluated along with the original FV57. The results indicated that the mismatched disulfide bond of FV57 linking the light chain FR1 and CDR3 would confer deleterious negative effects on its activity against RV, likely due to spatial hindrance in the light chain CDR3. Moreover, avoidance of the disulfide bond mismatch provided an additional 30% protective efficacy against RV infection in the mouse RV challenge model. Thus, modifications of FV57 to eliminate the disulfide bond mismatch may provide a candidate therapeutic agent for effective PEP against rabies.
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Anticuerpos Neutralizantes/inmunología , Antígenos Virales/inmunología , Glicoproteínas/inmunología , Profilaxis Posexposición , Vacunas Antirrábicas/inmunología , Virus de la Rabia/inmunología , Rabia/prevención & control , Anticuerpos de Cadena Única/inmunología , Proteínas del Envoltorio Viral/inmunología , Animales , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/administración & dosificación , Anticuerpos Neutralizantes/genética , Anticuerpos Antivirales/administración & dosificación , Anticuerpos Antivirales/genética , Anticuerpos Antivirales/inmunología , Línea Celular , Cricetinae , Cisteína/genética , Femenino , Ratones , Mutación , Rabia/inmunología , Vacunas Antirrábicas/administración & dosificación , Vacunas Antirrábicas/genética , Anticuerpos de Cadena Única/administración & dosificación , Anticuerpos de Cadena Única/genéticaRESUMEN
Rabies virus (RABV) causes a fatal infectious disease, but effective protection may be achieved with the use of rabies immunoglobulin and a rabies vaccine. Virus-neutralizing antibodies (VNA), which play an important role in the prevention of rabies, are commonly evaluated by the RABV neutralizing test. For determining serum VNA levels or virus titers during the RABV vaccine manufacturing process, reliability of the assay method is highly important and mainly dependent on the diagnostic antibody. Most diagnostic antibodies are monoclonal antibodies (mAbs) made from hybridoma cell lines and are costly and time consuming to prepare. Thus, production of a cost-effective mAb for determining rabies VNA levels or RABV titers is needed. In this report, we describe the prokaryotic production of a RABV-specific single-chain variable fragment (scFv) protein with a His-tag (scFv98H) from a previously constructed plasmid in a bioreactor, including the purification and refolding process as well as the functional testing of the protein. The antigen-specific binding characteristics, affinity, and relative affinity of the purified protein were tested. The scFv98H antibody was compared with a commercial RABV nucleoprotein mAb for assaying the VNA level of anti-rabies serum samples from different sources or testing the growth kinetics of RABV strains for vaccine manufactured in China. The results indicated that scFv98H may be used as a novel diagnostic tool to assay VNA levels or virus titers and may be used as an alternative for the diagnostic antibody presently employed for these purposes.
Asunto(s)
Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/metabolismo , Glicoproteínas/inmunología , Vacunas Antirrábicas/inmunología , Virus de la Rabia/inmunología , Virus de la Rabia/metabolismo , Anticuerpos de Cadena Única/biosíntesis , Proteínas Virales/inmunologíaRESUMEN
Rabies is a fatal infectious disease requiring efficient protection provided by post-exposure prophylaxis (PEP) with rabies immunoglobulin (RIG). The single-chain Fv fragment (scFv) is a small engineered antigen binding protein derived from antibody variable heavy (V(H)) and light (V(L)) chains. This novel antibody format may potentially replace the current application of RIG to detect and neutralize rabies virus (RV). However, the broad use of scFvs is confined by their generally low stability. In this study, a scFv (FV57) was constructed based on the monoclonal antibody, MAB57, against RV. To enhance its stability and neutralizing potency, a disulfide-stabilized scFv, ds-FV57, was also derived by introduction of cysteines at V(H)44 and V(L)100. Furthermore, the cysteine at V(L)85 of ds-FV57 was mutated to serine to construct ds-FV57(VL85Ser) in order to avoid potential mis-formed disulfide bonds which would alter the affinity of the scFv. The stability and activity of all three proteins expressed in Escherichia coli were evaluated. All of the constructed scFvs could provide efficient protection against RV infection both in vivo and in vitro. However, the stability of ds-FV57(VL85Ser) was notably improved, and its in vitro neutralizing potency against RV infection was enhanced. Our findings from these stabilization modifications support the feasibility of developing scFvs for PEP treatment of rabies.