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Vanillic acid (VA) has shown antioxidant and anti-inflammatory activities in different cell types, but its biological effects in the context of early embryo development have not yet been clarified. In the current study, the impact of VA supplementation during in vitro maturation (IVM) and/or post-fertilization (in vitro culture; IVC) on redox homeostasis, mitochondrial function, AKT signaling, developmental competence, and the quality of bovine pre-implantation embryos was investigated. The results showed that dual exposure to VA during IVM and late embryo culture (IVC3) significantly improved the blastocyst development rate, reduced oxidative stress, and promoted fatty acid oxidation as well as mitochondrial activity. Additionally, the total numbers of cells and trophectoderm cells per blastocyst were higher in the VA-treated group compared to control (p < 0.05). The RT-qPCR results showed down-regulation of the mRNA of the apoptosis-specific markers and up-regulation of AKT2 and the redox homeostasis-related gene TXN in the treated group. Additionally, the immunofluorescence analysis showed high levels of pAKT-Ser473 and the fatty acid metabolism marker CPT1A in embryos developed following VA treatment. In conclusion, the study reports, for the first time, the embryotrophic effects of VA, and the potential linkage to AKT signaling pathway that could be used as an efficacious protocol in assisted reproductive technologies (ART) to improve human fertility.
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Técnicas de Maturação in Vitro de Oócitos , Oócitos , Animais , Bovinos , Humanos , Oócitos/metabolismo , Técnicas de Maturação in Vitro de Oócitos/métodos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ácido Vanílico/farmacologia , Estresse Oxidativo , Desenvolvimento Embrionário , Transdução de Sinais , Ácidos Graxos/metabolismoRESUMO
Melatonin, an antioxidant hormone secreted by the pineal gland, has been recognized as a regulator for numerous biological events. The deleterious effects of juglone, a polyphenolic extract of walnut trees, on embryo development has been previously reported. In the current study, we aimed to display the impact of melatonin administrated during in vitro oocyte maturation (IVM) on juglone-treated oocytes. Thus, in vitro matured oocytes were collected after 24 h post incubation with juglone in the presence or absence of melatonin. Reactive oxygen species (ROS), glutathione (GSH) content, mitochondrial distribution, and the relative abundance of mRNA transcription levels were assessed in oocytes, in addition, oocytes were in vitro fertilized to check the competency levels of oocytes to generate embryos. We found that administration of melatonin during the maturation of oocytes under juglone stress significantly improved the cleavage rate, 8-16 cell-stage embryos and day-8 blastocysts when compared to the sole juglone treatment. In addition, the fluorescence intensity of ROS increased, whereas the GSH decreased in juglone-treated oocytes compared to melatonin-juglone co-treated and untreated ones. Additionally, a significant increase in the mitochondrial aberrant pattern, the pattern that was normalized following melatonin supplementation, was observed following juglone administration. The mRNA analysis using RT-qPCR revealed a significant upregulation of autophagy and oxidative-stress-specific markers in the juglone-treated group compared to the co-treatment and control. In conclusion, the study reveals, for the first time, a protective effect of melatonin against the oxidative stress initiated following juglone treatment during the in vitro maturation of oocytes.
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Background & Aims: Elimination of chronic HBV/HDV infection remains a major global health challenge. Targeting excessive hepatitis B surface antigen (HBsAg) release may provide an interesting window of opportunity to break immune tolerance and to achieve a functional cure using additional antivirals. Methods: We evaluated a HBsAg-specific human monoclonal antibody, as part of either a prophylactic or therapeutic strategy, against HBV/HDV infection in cell culture models and in human-liver chimeric mice. To assess prophylactic efficacy, mice were passively immunized prior to infection with HBV or HBV/HDV (coinfection and superinfection setting). Therapeutic efficacy was assessed in HBV and HBV/HDV-coinfected mice receiving 4 weeks of treatment. Viral parameters (HBV DNA, HDV RNA and HBsAg) were assessed in mouse plasma. Results: The antibody could effectively prevent HBV/HDV infection in a dose-dependent manner with IC50 values of â¼3.5 ng/ml. Passive immunization showed complete protection of mice from both HBV and HBV/HDV coinfection. Moreover, HDV superinfection was either completely prevented or at least attenuated in HBV-infected mice. Finally, antibody treatment in mice with established HBV/HDV infection resulted in a significant decline in viremia and a concomitant drop in on-treatment HBsAg, with a moderate viral rebound following treatment cessation. Conclusion: We present data on a valuable antibody candidate that could complement other antivirals in strategies aimed at achieving functional cure of chronic HBV and HDV infection. Impact and implications: Patients chronically infected with HBV may eventually develop liver cancer and are at great risk of being superinfected with HDV, which worsens and accelerates disease progression. Unfortunately, current treatments can rarely eliminate both viruses from chronically infected patients. In this study, we present data on a novel antibody that is able to prevent chronic HBV/HDV infection in a mouse model with a humanized liver. Moreover, antibody treatment of HBV/HDV-infected mice strongly diminishes viral loads during therapy. This antibody is a valuable candidate for further clinical development.
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The World Health Organization (WHO) recently alerted the emergence of new pathogens causing acute hepatitis in children across several countries. This new situation directs us to the screening of neglected pathogens that cause acute hepatitis. Q-fever is a zoonotic disease, caused by Coxiella burnetii. Although a high seroprevalence of Coxiella burnetii was recorded in animals present in Egypt, Q-fever is still a neglected disease, and the diagnosis of Q-fever is not routinely performed in Egyptian hospitals. In this study, we performed a retrospective assessment for Coxiella burnetii in cases of hepatitis of unknown causes (HUC) enrolled in Assiut University hospitals, in Egypt. Out of 64 samples of HUC, 54 samples were negative for all hepatitis markers, labeled as acute hepatitis of unknown etiology (AHUE), and 10 samples tested positive for adenovirus and Hepatitis E virus (HEV). Q-fever was detected in 3 out of 54 (5.6%) of AHUE, and one sample was confirmed as coinfection of HEV/Q-fever. Jaundice was the most common clinical symptom developed in the patients. In conclusion, Coxiella burnetii was found to be a potential cause of acute hepatitis in HUC. The diagnosis of Q-fever should be considered in acute hepatitis cases in Egyptian hospitals.
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Juglone, a major naphthalenedione component of walnut trees, has long been used in traditional medicine as an antimicrobial and antitumor agent. Nonetheless, its impact on oocyte and preimplantation embryo development has not been entirely clarified. Using the bovine model, we sought to elucidate the impact of juglone treatment during the in vitro maturation (IVM) of oocytes on their maturation and development of embryos. Results showed a severe reduction in oocyte nuclear maturation and cumulus expansion and a significant increase in mitochondrial dysfunction and reactive oxygen species (ROS) levels in cumulus-oocyte complexes (COCs) treated with juglone (12.5, 25.0, and 50.0 µM). In addition, RT-qPCR showed downregulation of the expansion-related (HAS2, TNFAIP6, PTX3, and PTGS2) and mitochondrial (ATPase6 and ATP5F1E) genes in juglone-treated COCs. Moreover, the development rates of day 4 total cleavage and 8-16 cell stage embryos, as well as day 8 blastocysts, were significantly reduced following exposure to juglone. Using immunofluorescence, the apoptotic marker caspase-9 was overexpressed in oocytes exposed to juglone (25.0 µM) compared to the untreated control. In conclusion, our study reports that exposing bovine oocytes to 12.5-50.0 µM of juglone can reduce their development through the direct induction of ROS accumulation, apoptosis, and mitochondrial dysfunction.
Assuntos
Apoptose , Embrião de Mamíferos/patologia , Mitocôndrias/patologia , Naftoquinonas/toxicidade , Oócitos/patologia , Estresse Oxidativo/efeitos dos fármacos , Animais , Blastocisto/efeitos dos fármacos , Blastocisto/patologia , Bovinos , Citotoxinas/toxicidade , Embrião de Mamíferos/efeitos dos fármacos , Desenvolvimento Embrionário , Feminino , Técnicas de Maturação in Vitro de Oócitos/métodos , Mitocôndrias/efeitos dos fármacos , Oócitos/efeitos dos fármacos , Gravidez , Espécies Reativas de Oxigênio/metabolismoRESUMO
Nicotinamide (NAM), the amide form of vitamin B3, plays pivotal roles in regulating various cellular processes including energy production and maintenance of genomic stability. The current study aimed at deciphering the effect of NAM, when administered during in vitro maturation (IVM), on the developmental competence of bovine preimplantation embryos. Our results showed that low NAM concentrations reduced the oxidative stress and improved mitochondrial profile, total cleavage and 8-16 cell stage embryo development whereas the opposite profile was observed upon exposure to high NAM concentrations (10 mM onward). Remarkably, the hatching rates of day-7 and day-8 blastocysts were significantly improved under 0.1 mM NAM treatment. Using RT-qPCR and immunofluorescence, the autophagy-related (Beclin-1 (BECN1), LC3B, and ATG5) and the apoptotic (Caspases; CASP3 and 9) markers were upregulated in oocytes exposed to high NAM concentration (40 mM), whereas only CASP3 was affected, downregulated, following 0.1 mM treatment. Additionally, the number of cells per blastocyst and the levels of SIRT1, PI3K, AKT, and mTOR were higher, while the inner cell mass-specific transcription factors GATA6, SOX2, and OCT4 were more abundant, in day-8 embryos of NAM-treated group. Taken together, to our knowledge, this is the first study reporting that administration of low NAM concentrations during IVM can ameliorate the developmental competence of embryos through the potential regulation of oxidative stress, apoptosis, and SIRT1/AKT signaling.
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Blastocisto/metabolismo , Técnicas de Maturação in Vitro de Oócitos/métodos , Niacinamida/uso terapêutico , Oócitos/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Bovinos , Suplementos Nutricionais , Feminino , Humanos , Transdução de SinaisRESUMO
Melatonin, a nighttime-secreted antioxidant hormone produced by the pineal gland, and AKT, a serine/threonine-specific protein kinase, have been identified as regulators for several cellular processes essential for reproduction. The current study aimed to investigate the potential interplay between melatonin and AKT in bovine oocytes in the context of embryo development. Results showed that the inclusion of SH6, a specific AKT inhibitor, during in vitro maturation (IVM) significantly reduced oocyte maturation, cumulus cell expansion, cleavage, and blastocyst development that were rescued upon addition of melatonin. Oocytes treated with SH6 in the presence of melatonin showed lower levels of reactive oxygen species (ROS) and blastocysts developed exhibited low apoptosis while the mitochondrial profile was significantly improved compared to the SH6-treated group. The RT-qPCR results showed up-regulation of the mRNA of maturation-, mitochondrial-, and cumulus expansion-related genes including GDF-9, BMP-15, MARF1, ATPase, ATP5F1E, POLG2, HAS2, TNFAIP6, and PTGS2 and down-regulation of Bcl-2 associated X apoptosis regulator (BAX), caspase 3, and p21 involved in apoptosis and cell cycle arrest in melatonin-SH6 co-treated group compared to SH6 sole treatment. The immunofluorescence showed high levels of caspase 3 and caspase 9, and low AKT phosphorylation in the SH6-treated group compared to the control and melatonin-SH6 co-treatment. Taken together, our results showed the importance of both melatonin and AKT for overall embryonic developmental processes and, for the first time, we report that melatonin could neutralize the deleterious consequences of AKT inhibition, suggesting a potential role in regulation of AKT signaling in bovine oocytes.
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Desenvolvimento Embrionário/efeitos dos fármacos , Melatonina/farmacologia , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Animais , Apoptose/efeitos dos fármacos , Bovinos , Núcleo Celular/metabolismo , Células do Cúmulo/efeitos dos fármacos , Feminino , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
In the hepatitis C virus (HCV) envelope glycoproteins E1 and E2, which form a heterodimer, E2 is the receptor binding protein and the major target of neutralizing antibodies, whereas the function of E1 remains less characterized. To investigate E1 functions, we generated a series of mutants in the conserved residues of the C-terminal region of the E1 ectodomain in the context of an infectious clone. We focused our analyses on two regions of interest. The first region is located in the middle of the E1 glycoprotein (between amino acid [aa] 270 and aa 291), which contains a conserved hydrophobic sequence and was proposed to constitute a putative fusion peptide. The second series of mutants was generated in the region from aa 314 to aa 342 (the aa314-342 region), which has been shown to contain two α helices (α2 and α3) by nuclear magnetic resonance studies. Of the 22 generated mutants, 20 were either attenuated or noninfectious. Several mutations modulated the virus's dependence on claudin-1 and the scavenger receptor BI coreceptors for entry. Most of the mutations in the putative fusion peptide region affected virus assembly. Conversely, mutations in the α-helix aa 315 to 324 (315-324) residues M318, W320, D321, and M322 resulted in a complete loss of infectivity without any impact on E1E2 folding and on viral assembly. Further characterization of the W320A mutant in the HCVpp model indicated that the loss of infectivity was due to a defect in viral entry. Together, these results support a role for E1 in modulating HCV interaction with its coreceptors and in HCV assembly. They also highlight the involvement of α-helix 315-324 in a late step of HCV entry.IMPORTANCE HCV is a major public health problem worldwide. The virion harbors two envelope proteins, E1 and E2, which are involved at different steps of the viral life cycle. Whereas E2 has been extensively characterized, the function of E1 remains poorly defined. We characterized here the function of the putative fusion peptide and the region containing α helices of the E1 ectodomain, which had been previously suggested to be important for virus entry. We could confirm the importance of these regions for the virus infectivity. Interestingly, we found several residues modulating the virus's dependence on several HCV receptors, thus highlighting the role of E1 in the interaction of the virus with cellular receptors. Whereas mutations in the putative fusion peptide affected HCV infectivity and morphogenesis, several mutations in the α2-helix region led to a loss of infectivity with no effect on assembly, indicating a role of this region in virus entry.
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Hepacivirus/fisiologia , Proteínas do Envelope Viral/metabolismo , Montagem de Vírus , Internalização do Vírus , Linhagem Celular , Análise Mutacional de DNA , Hepatócitos/virologia , Humanos , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Proteínas do Envelope Viral/genéticaRESUMO
Monoclonal antibodies (mAbs) targeting the hepatitis C virus (HCV) envelope have been raised mainly against envelope protein 2 (E2), while the antigenic epitopes of envelope protein 1 (E1) are not fully identified. Here we describe the detailed characterization of a human mAb, designated A6, generated from an HCV genotype 1b infected patient. ELISA results showed reactivity of mAb A6 to full-length HCV E1E2 of genotypes 1a, 1b and 2a. Epitope mapping identified a region spanning amino acids 230-239 within the N-terminal region of E1 as critical for binding. Antibody binding to this epitope was not conformation dependent. Neutralization assays showed that mAb A6 lacks neutralizing capacity and does not interfere with the activity of known neutralizing antibodies. In summary, mAb A6 is an important tool to study the structure and function of E1 within the viral envelope, a crucial step in the development of an effective prophylactic HCV vaccine.
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Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Hepacivirus/imunologia , Hepatite C/imunologia , Proteínas do Envelope Viral/imunologia , Anticorpos Monoclonais/análise , Anticorpos Neutralizantes/análise , Mapeamento de Epitopos , Genótipo , Hepacivirus/química , Hepacivirus/genética , Hepatite C/genética , Humanos , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genéticaRESUMO
Infections with hepatitis C virus (HCV) represent a worldwide health burden and a prophylactic vaccine is still not available. Liver transplantation (LT) is often the only option for patients with HCV-induced end-stage liver disease. However, immediately after transplantation, the liver graft becomes infected by circulating virus, resulting in accelerated progression of liver disease. Although the efficacy of HCV treatment using direct-acting antivirals has improved significantly, immune compromised LT-patients and patients with advanced liver disease remain difficult to treat. As an alternative approach, interfering with viral entry could prevent infection of the donor liver. We generated a human monoclonal antibody (mAb), designated 2A5, which targets the HCV envelope. The neutralizing activity of mAb 2A5 was assessed using multiple prototype and patient-derived HCV pseudoparticles (HCVpp), cell culture produced HCV (HCVcc), and a human-liver chimeric mouse model. Neutralization levels observed for mAb 2A5 were generally high and mostly superior to those obtained with AP33, a well-characterized HCV-neutralizing monoclonal antibody. Using humanized mice, complete protection was observed after genotype 1a and 4a HCV challenge, while only partial protection was achieved using gt1b and 6a isolates. Epitope mapping revealed that mAb 2A5 binding is conformation-dependent and identified the E2-region spanning amino acids 434 to 446 (epitope II) as the predominant contact domain. CONCLUSION: mAb 2A5 shows potent anti-HCV neutralizing activity both in vitro and in vivo and could hence represent a valuable candidate to prevent HCV recurrence in LT-patients. In addition, the detailed identification of the neutralizing epitope can be applied for the design of prophylactic HCV vaccines.
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Anticorpos Neutralizantes/farmacologia , Anticorpos Neutralizantes/uso terapêutico , Hepacivirus/efeitos dos fármacos , Anticorpos Anti-Hepatite C/farmacologia , Anticorpos Anti-Hepatite C/uso terapêutico , Hepatite C/tratamento farmacológico , Proteínas do Envelope Viral/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Anticorpos Neutralizantes/imunologia , Modelos Animais de Doenças , Mapeamento de Epitopos , Epitopos/genética , Epitopos/imunologia , Genótipo , Hepacivirus/genética , Hepacivirus/imunologia , Hepatite C/imunologia , Hepatite C/prevenção & controle , Hepatite C/virologia , Anticorpos Anti-Hepatite C/imunologia , Humanos , Transplante de Fígado , Camundongos , Camundongos SCID , Mutação , Testes de Neutralização , Relação Estrutura-Atividade , Proteínas do Envelope Viral/genética , Internalização do Vírus/efeitos dos fármacosRESUMO
UNLABELLED: Aminoquinolines and piperazines, linked or not, have been used successfully to treat malaria, and some molecules of this family also exhibit antiviral properties. Here we tested several derivatives of 4-aminoquinolines and piperazines for their activity against hepatitis C virus (HCV). We screened 11 molecules from three different families of compounds, and we identified anti-HCV activity in cell culture for six of them. Of these, we selected a compound (B5) that is currently ending clinical phase I evaluation for neurodegenerative diseases. In hepatoma cells, B5 inhibited HCV infection in a pangenotypic and dose-dependent manner, and its antiviral activity was confirmed in primary hepatocytes. B5 also inhibited infection by pseudoparticles expressing HCV envelope glycoproteins E1 and E2, and we demonstrated that it affects a postattachment stage of the entry step. Virus with resistance to B5 was selected by sequential passage in the presence of the drug, and reverse genetics experiments indicated that resistance was conferred mainly by a single mutation in the putative fusion peptide of E1 envelope glycoprotein (F291I). Furthermore, analyses of the effects of other closely related compounds on the B5-resistant mutant suggest that B5 shares a mode of action with other 4-aminoquinoline-based molecules. Finally, mice with humanized liver that were treated with B5 showed a delay in the kinetics of the viral infection. In conclusion, B5 is a novel interesting anti-HCV molecule that could be used to decipher the early steps of the HCV life cycle. IMPORTANCE: In the last 4 years, HCV therapy has been profoundly improved with the approval of direct-acting antivirals in clinical practice. Nevertheless, the high costs of these drugs limit access to therapy in most countries. The present study reports the identification and characterization of a compound (B5) that inhibits HCV propagation in cell culture and is currently ending clinical phase I evaluation for neurodegenerative diseases. This molecule inhibits the HCV life cycle by blocking virus entry. Interestingly, after selection of drug-resistant virus, a resistance mutation in the putative fusion peptide of E1 envelope glycoprotein was identified, indicating that B5 could be used to further investigate the fusion mechanism. Furthermore, mice with humanized liver treated with B5 showed a delay in the kinetics of the viral infection. In conclusion, B5 is a novel interesting anti-HCV molecule that could be used to decipher the early steps of the HCV life cycle.
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Aminoquinolinas/farmacologia , Antivirais/farmacologia , Hepacivirus/efeitos dos fármacos , Aminoquinolinas/química , Aminoquinolinas/isolamento & purificação , Animais , Antivirais/química , Antivirais/isolamento & purificação , Células Cultivadas , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Farmacorresistência Viral , Hepatite C/tratamento farmacológico , Hepatócitos/virologia , Humanos , Camundongos , Camundongos SCID , Modelos Moleculares , Estrutura Molecular , Mutação de Sentido Incorreto , Genética Reversa , Resultado do Tratamento , Proteínas do Envelope Viral/genética , Internalização do Vírus/efeitos dos fármacosRESUMO
UNLABELLED: Despite the validation of direct-acting antivirals for hepatitis C treatment, the discovery of new compounds with different modes of action may still be of importance for the treatment of special patient populations. We recently identified a natural molecule, epigallocatechin-3-gallate (EGCG), as an inhibitor of hepatitis C virus (HCV) targeting the viral particle. The aim of this work was to discover new natural compounds with higher anti-HCV activity than that of EGCG and determine their mode of action. Eight natural molecules with structure similarity to EGCG were selected. HCV JFH1 in cell culture and HCV pseudoparticle systems were used to determine the antiviral activity and mechanism of action of the compounds. We identified delphinidin, a polyphenol belonging to the anthocyanidin family, as a new inhibitor of HCV entry. Delphinidin inhibits HCV entry in a pangenotypic manner by acting directly on the viral particle and impairing its attachment to the cell surface. Importantly, it is also active against HCV in primary human hepatocytes, with no apparent cytotoxicity and in combination with interferon and boceprevir in cell culture. Different approaches showed that neither aggregation nor destruction of the particle occurred. Cryo-transmission electron microscopy observations of HCV pseudoparticles treated with delphinidin or EGCG showed a bulge on particles that was not observed under control conditions. In conclusion, EGCG and delphinidin inhibit HCV entry by a new mechanism, i.e., alteration of the viral particle structure that impairs its attachment to the cell surface. IMPORTANCE: In this article, we identify a new inhibitor of hepatitis C virus (HCV) infection, delphinidin, that prevents HCV entry. This natural compound, a plant pigment responsible for the blue-purple color of flowers and berries, belongs to the flavonoid family, like the catechin EGCG, the major component present in green tea extract, which is also an inhibitor of HCV entry. We studied the mode of action of these two compounds against HCV and demonstrated that they both act directly on the virus, inducing a bulging of the viral envelope. This deformation might be responsible for the observed inhibition of virus attachment to the cell surface. The discovery of such HCV inhibitors with an unusual mode of action is important to better characterize the mechanism of HCV entry into hepatocytes and to help develop a new class of HCV entry inhibitors.
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Antivirais/farmacologia , Hepacivirus/efeitos dos fármacos , Hepacivirus/fisiologia , Polifenóis/farmacologia , Internalização do Vírus/efeitos dos fármacos , Antocianinas/administração & dosagem , Antocianinas/farmacologia , Antivirais/administração & dosagem , Catequina/análogos & derivados , Catequina/farmacologia , Linhagem Celular , Microscopia Crioeletrônica , Avaliação Pré-Clínica de Medicamentos , Células HEK293 , Hepacivirus/ultraestrutura , Hepatócitos/efeitos dos fármacos , Hepatócitos/virologia , Humanos , Interferon-alfa/administração & dosagem , Polifenóis/administração & dosagem , Prolina/administração & dosagem , Prolina/análogos & derivadosRESUMO
UNLABELLED: Hepatitis C virus (HCV)-induced endstage liver disease is currently a major indication for liver transplantation. After transplantation the donor liver inevitably becomes infected with the circulating virus. Monoclonal antibodies (mAbs) against the HCV coreceptor scavenger receptor class B type I (SR-BI) inhibit HCV infection of different genotypes, both in cell culture and in humanized mice. Anti-SR-BI mAb therapy is successful even when initiated several days after HCV exposure, supporting its potential applicability to prevent HCV reinfection of liver allografts. However, HCV variants with reduced SR-BI dependency have been described in the literature, which could potentially limit the use of SR-BI targeting therapy. In this study we show, both in a preventative and postexposure setting, that humanized mice infected with HCV variants exhibiting increased in vitro resistance to SR-BI-targeting molecules remain responsive to anti-SR-BI mAb therapy in vivo. A 2-week antibody therapy readily cleared HCV RNA from the circulation of infected humanized mice. We found no evidence supporting increased SR-BI-receptor dependency of viral particles isolated from humanized mice compared to cell culture-produced virus. However, we observed that, unlike wild-type virus, the in vitro infectivity of the resistant variants was inhibited by both human high density lipoprotein (HDL) and very low density lipoprotein (VLDL). The combination of mAb1671 with these lipoproteins further increased the antiviral effect. CONCLUSION: HCV variants that are less dependent on SR-BI in vitro can still be efficiently blocked by an anti-SR-BI mAb in humanized mice. Since these variants are also more susceptible to neutralization by anti-HCV envelope antibodies, their chance of emerging during anti-SR-BI therapy is severely reduced. Our data indicate that anti-SR-BI receptor therapy could be an effective way to prevent HCV infection in a liver transplant setting.
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Anticorpos Monoclonais/uso terapêutico , Hepatite C/tratamento farmacológico , Receptores Depuradores Classe B/imunologia , Animais , Linhagem Celular Tumoral , Hepacivirus/efeitos dos fármacos , Hepacivirus/patogenicidade , Hepatite C/virologia , Humanos , Lipoproteínas/farmacologia , Lipoproteínas/uso terapêutico , Camundongos SCID , Resultado do Tratamento , Ácidos Tri-IodobenzoicosRESUMO
The role of the NS3 protease in HCV replication was demonstrated by the ability of a protease inhibitor cocktail (10 microg/ml) to abolish the induced cytopathic effect in RAW macrophages upon infection with Egyptian sera. The HCV protease gene was amplified from Egyptian sera by nested PCR and cloned downstream of the CMV promotor in a mammalian expression plasmid, which was then used to transform bacteria. Colonies carrying the gene in the correct orientation were subjected to large-scale plasmid purification followed by sequencing. Phylogenetic comparison of the sequence obtained with published sequences from different genotypes confirmed that our sequence belongs to genotype 4a. Of the other genotypes, the most closely related ones were from genotype 1. Multiple alignments of protease peptides showed that the catalytic triads and binding residues for substrate, Zn2+ and the NS4 cofactor are conserved among different isolates, including ours, and confirmed the closer homology between NS3 of genotypes 4 and 1. The HCV-protease-encoding construct was successfully transcribed in both mammalian cells and mice. Mouse antibodies produced against the protease-encoding-construct detected the 18-kDa enzyme in lysates of cells transfected with the construct by Western blotting, and in the media of infected cells by ELISA.