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1.
Virulence ; 12(1): 2430-2442, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34517779

RESUMO

Severe acute respiratory syndrome CoV-2 (SARS-CoV-2) is currently causing a worldwide threat with its unusually high transmission rates and rapid evolution into diverse strains. Unlike typical respiratory viruses, SARS-CoV-2 frequently causes systemic infection by breaking the boundaries of the respiratory systems. The development of animal models recapitulating the clinical manifestations of COVID-19 is of utmost importance not only for the development of vaccines and antivirals but also for understanding the pathogenesis. However, there has not been developed an animal model for systemic infection of SARS-CoV-2 representing most aspects of the clinical manifestations of COVID-19 with systemic symptoms. Here we report that a Roborovski hamster strain SH101, a laboratory inbred hamster strain of P. roborovskii, displayed most symptoms of systemic infection upon SARS-CoV-2 infection as in the case of the human counterpart, unlike current COVID-19 animal models. Roborovski hamster strain SH101 post-infection of SARS-CoV-2 represented most clinical symptoms of COVID-19 such as snuffling, labored breathing, dyspnea, cough, hunched posture, progressive weight loss, ruffled fur, and high fever following shaking chills. Histological examinations also revealed initial right-predominated pneumonia as well as slight organ damages in the brain and liver, manifesting systemic COVID-19 cases. Considering the merit of a small animal as well as its clinical manifestations of SARS-CoV-2 infection in human, this hamster model seems to provide an ideal tool to investigate COVID-19.


Assuntos
COVID-19 , Cricetinae/classificação , Modelos Animais de Doenças , SARS-CoV-2 , Animais , Temperatura Corporal , Encéfalo/patologia , COVID-19/patologia , COVID-19/fisiopatologia , Feminino , Fígado/patologia , Pulmão/patologia , Masculino , Mesocricetus , Camundongos , Camundongos Transgênicos
2.
Curr Protoc ; 1(9): e236, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34491634

RESUMO

Human artificial chromosomes (HACs) are considered promising tools for gene delivery, functional analyses, and gene therapy. HACs have the potential to overcome many of the problems caused by the use of viral-based gene transfer systems, such as limited cloning capacity, lack of copy number control, and insertional mutagenesis during integration into host chromosomes. The recently developed alphoidtetO -HAC has an advantage over other HAC vectors because it can be easily eliminated from dividing cells by inactivation of its conditional kinetochore. This provides a unique control mechanism to study phenotypes induced by a gene or genes carried on the HAC. The alphoidtetO -HAC has a single gene acceptor loxP site that allows insertion of an individual gene of interest or a cluster of genes of up to several Mb in size in Chinese hamster ovary (CHO) hybrid cells. The HACs carrying chromosomal copies of genes can then be transferred from these donor CHO cells to different recipient cells of interest via microcell-mediated chromosome transfer (MMCT). Here, we describe a detailed protocol for loading a gene of interest into the alphoidtetO -HAC vector and for the subsequent transfer of the HAC to recipient cells using an improved MMCT protocol. The original MMCT protocol includes treatment of donor cells with colcemid to induce micronucleation, wherein the HAC becomes surrounded with a nuclear membrane. That step is followed by disarrangement of the actin cytoskeleton using cytochalasin B to help induce microcell formation. The updated MMCT protocol, described here, features the replacement of colcemid and cytochalasin B with TN16 + griseofulvin and latrunculin B, respectively, and the use of collagen/laminin surface coating to promote attachment of metaphase cells to plates during micronuclei induction. These modifications increase the efficiency of HAC transfer to recipient cells ten fold. The improved MMCT protocol has been successfully tested on several recipient cell lines, including human mesenchymal stem cells and mouse embryonic stem cells. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Insertion of a BAC containing a gene of interest into a single loxP loading site of alphoidtetO -HAC in hamster CHO cells Basic Protocol 2: Microcell-mediated chromosome transfer from donor hamster CHO cells to mammalian cells.


Assuntos
Cromossomos Artificiais Humanos , Animais , Células CHO , Cromossomos Artificiais Humanos/genética , Cricetinae , Cricetulus , Técnicas de Transferência de Genes , Genômica , Humanos , Camundongos
3.
Braz J Biol ; 83: e242439, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34468507

RESUMO

Plinia cauliflora (Mart.) Kausel, popularly known as jabuticaba, is rich in polyphenols. Phenolic compounds exhibit several biological properties, which reflect on biomarkers such as biochemical parameters. In the present study, we evaluated the plasmatic levels of glucose, total cholesterol, HDL-cholesterol, triglycerides, and uric acid of Chinese hamsters fed for 45 days with a regular diet or cholesterol-enriched diet supplemented with a liquid extract obtained from P. cauliflora fruits residues standardized in ellagic acid and total phenolic compounds. The results showed that the concentrated extract obtained from jabuticaba residues increased the glycemia of animals fed with a regular diet and reduced the plasmatic uric acid levels of animals fed with a cholesterol-enriched diet. Since hyperuricemia is considered to be a significant risk factor of metabolic disorders and the principal pathological basis of gout, the liquid extract from P. cauliflora fruits residues would be a promising candidate as a novel hypouricaemic agent for further investigation.


Assuntos
Frutas , Myrtaceae , Animais , Cricetinae , Cricetulus , Fenóis , Extratos Vegetais
4.
Vet Res ; 52(1): 121, 2021 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-34530902

RESUMO

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic is causing a global crisis. It is still unresolved. Although many therapies and vaccines are being studied, they are still in their infancy. As this pandemic continues, rapid and accurate research for the development of therapies and vaccines is needed. Therefore, it is necessary to understand characteristics of diseases caused by SARS-CoV-2 through animal models. Syrian hamsters are known to be susceptible to SARS-CoV-2. They were intranasally inoculated with SARS-CoV-2. At 2, 4, 8, 12, and 16 days post-infection (dpi), these hamsters were euthanized, and tissues were collected for ultrastructural and microstructural examinations. Microscopic lesions were prominent in the upper and lower respiratory tracts from 2 and 4 dpi groups, respectively. The respiratory epithelium in the trachea, bronchiole, and alveolar showed pathological changes. Inflammatory cells including neutrophils, lymphocytes, macrophages, and eosinophils were infiltrated in/around tracheal lamina propria, pulmonary vessels, alveoli, and bronchiole. In pulmonary lesions, alveolar wall was thickened with infiltrated inflammatory cells, mainly neutrophils and macrophages. In the trachea, epithelial damages started from 2 dpi and recovered from 8 dpi, consistent with microscopic results, High levels of SARS-CoV-2 nucleoprotein were detected at 2 dpi and 4 dpi. In the lung, lesions were most severe at 8 dpi. Meanwhile, high levels of SARS-CoV-2 were detected at 4 dpi. Electron microscopic examinations revealed cellular changes in the trachea epithelium and alveolar epithelium such as vacuolation, sparse micro-organelle, and poor cellular margin. In the trachea epithelium, the number of cytoplasmic organelles was diminished, and small vesicles were prominent from 2 dpi. Some of these electron-lucent vesicles were filled with virion particles. From 8 dpi, the trachea epithelium started to recover. Because of shrunken nucleus and swollen cytoplasm, the N/C ratio of type 2 pneumocyte decreased at 8 and 12 dpi. From 8 dpi, lamellar bodies on type 2 pneumocyte cytoplasm were increasingly observed. Their number then decreased from 16 dpi. However, there was no significant change in type 1 pneumocyte. Viral vesicles were only observed in the cytoplasm of type 2 pneumocyte. In conclusion, ultra- and micro-structural changes presented in this study may provide useful information for SARS-CoV-2 studies in various fields.


Assuntos
COVID-19/patologia , Sistema Respiratório/patologia , SARS-CoV-2/patogenicidade , Animais , Cricetinae , Imuno-Histoquímica/veterinária , Masculino , Mesocricetus , Projetos Piloto , RNA Viral/química , RNA Viral/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Sistema Respiratório/química , Sistema Respiratório/ultraestrutura , Sistema Respiratório/virologia , Fatores de Tempo , Traqueia/patologia , Traqueia/ultraestrutura , Traqueia/virologia , Perda de Peso
5.
Nat Commun ; 12(1): 4869, 2021 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-34381043

RESUMO

In COVID-19, immune responses are key in determining disease severity. However, cellular mechanisms at the onset of inflammatory lung injury in SARS-CoV-2 infection, particularly involving endothelial cells, remain ill-defined. Using Syrian hamsters as a model for moderate COVID-19, we conduct a detailed longitudinal analysis of systemic and pulmonary cellular responses, and corroborate it with datasets from COVID-19 patients. Monocyte-derived macrophages in lungs exert the earliest and strongest transcriptional response to infection, including induction of pro-inflammatory genes, while epithelial cells show weak alterations. Without evidence for productive infection, endothelial cells react, depending on cell subtypes, by strong and early expression of anti-viral, pro-inflammatory, and T cell recruiting genes. Recruitment of cytotoxic T cells as well as emergence of IgM antibodies precede viral clearance at day 5 post infection. Investigating SARS-CoV-2 infected Syrian hamsters thus identifies cell type-specific effector functions, providing detailed insights into pathomechanisms of COVID-19 and informing therapeutic strategies.


Assuntos
COVID-19/imunologia , Modelos Animais de Doenças , Células Epiteliais Alveolares/imunologia , Animais , Cricetinae , Citocinas/genética , Citocinas/imunologia , Células Endoteliais/imunologia , Humanos , Imunoglobulina M/imunologia , Inflamação , Pulmão/imunologia , Macrófagos/imunologia , Mesocricetus , Monócitos/imunologia , SARS-CoV-2/imunologia , Transdução de Sinais , Linfócitos T Citotóxicos/imunologia , Receptores Toll-Like/imunologia
6.
Int J Mol Sci ; 22(16)2021 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-34445614

RESUMO

The anorexigenic neuropeptide prolactin-releasing peptide (PrRP) is involved in the regulation of food intake and energy expenditure. Lipidization of PrRP stabilizes the peptide, facilitates central effect after peripheral administration and increases its affinity for its receptor, GPR10, and for the neuropeptide FF (NPFF) receptor NPFF-R2. The two most potent palmitoylated analogs with anorectic effects in mice, palm11-PrRP31 and palm-PrRP31, were studied in vitro to determine their agonist/antagonist properties and mechanism of action on GPR10, NPFF-R2 and other potential off-target receptors related to energy homeostasis. Palmitoylation of both PrRP31 analogs increased the binding properties of PrRP31 to anorexigenic receptors GPR10 and NPFF-R2 and resulted in a high affinity for another NPFF receptor, NPFF-R1. Moreover, in CHO-K1 cells expressing GPR10, NPFF-R2 or NPFF-R1, palm11-PrRP and palm-PrRP significantly increased the phosphorylation of extracellular signal-regulated kinase (ERK), protein kinase B (Akt) and cAMP-responsive element-binding protein (CREB). Palm11-PrRP31, unlike palm-PrRP31, did not activate either c-Jun N-terminal kinase (JNK), p38, c-Jun, c-Fos or CREB pathways in cells expressing NPFF-1R. Palm-PrRP31 also has higher binding affinities for off-target receptors, namely, the ghrelin, opioid (KOR, MOR, DOR and OPR-L1) and neuropeptide Y (Y1, Y2 and Y5) receptors. Palm11-PrRP31 exhibited fewer off-target activities; therefore, it has a higher potential to be used as an anti-obesity drug with anorectic effects.


Assuntos
Cálcio/metabolismo , Lipoilação , Hormônio Liberador de Prolactina/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Neuropeptídeos/metabolismo , Animais , Células CHO , Cricetinae , Cricetulus , Humanos , Técnicas In Vitro , Hormônio Liberador de Prolactina/química , Hormônio Liberador de Prolactina/genética , Receptores Acoplados a Proteínas G/genética , Receptores de Neuropeptídeos/genética
7.
Int J Mol Sci ; 22(16)2021 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-34445625

RESUMO

Arthropod antimicrobial peptides (AMPs) offer a promising source of new leads to address the declining number of novel antibiotics and the increasing prevalence of multidrug-resistant bacterial pathogens. AMPs with potent activity against Gram-negative bacteria and distinct modes of action have been identified in insects and scorpions, allowing the discovery of AMP combinations with additive and/or synergistic effects. Here, we tested the synergistic activity of two AMPs, from the dung beetle Copris tripartitus (CopA3) and the scorpion Heterometrus petersii (Hp1090), against two strains of Escherichia coli. We also tested the antibacterial activity of two hybrid peptides generated by joining CopA3 and Hp1090 with linkers comprising two (InSco2) or six (InSco6) glycine residues. We found that CopA3 and Hp1090 acted synergistically against both bacterial strains, and the hybrid peptide InSco2 showed more potent bactericidal activity than the parental AMPs or InSco6. Molecular dynamics simulations revealed that the short linker stabilizes an N-terminal 310-helix in the hybrid peptide InSco2. This secondary structure forms from a coil region that interacts with phosphatidylethanolamine in the membrane bilayer model. The highest concentration of the hybrid peptides used in this study was associated with stronger hemolytic activity than equivalent concentrations of the parental AMPs. As observed for CopA3, the increasing concentration of InSco2 was also cytotoxic to BHK-21 cells. We conclude that AMP hybrids linked by glycine spacers display potent antibacterial activity and that the cytotoxic activity can be modulated by adjusting the nature of the linker peptide, thus offering a strategy to produce hybrid peptides as safe replacements or adjuncts for conventional antibiotic therapy.


Assuntos
Antibacterianos/farmacologia , Artrópodes/química , Bactérias/efeitos dos fármacos , Glicina/química , Hemólise/efeitos dos fármacos , Rim/efeitos dos fármacos , Proteínas Citotóxicas Formadoras de Poros/farmacologia , Animais , Antibacterianos/química , Apoptose , Células Cultivadas , Cricetinae , Camundongos , Proteínas Citotóxicas Formadoras de Poros/química
8.
Int J Mol Sci ; 22(16)2021 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-34445789

RESUMO

The SARS-CoV-2 pseudovirus is a commonly used strategy that mimics certain biological functions of the authentic virus by relying on biological legitimacy at the molecular level. Despite the fact that spike (S), envelope (E), and membrane (M) proteins together wrap up the SARS-CoV-2 virion, most of the reported pseudotype viruses consist of only the S protein. Here, we report that the presence of E and M increased the virion infectivity by promoting the S protein priming. The S, E, and M (SEM)-coated pseudovirion is spherical, containing crown-like spikes on the surface. Both S and SEM pseudoviruses packaged the same amounts of viral RNA, but the SEM virus bound more efficiently to cells stably expressing the viral receptor human angiotensin-converting enzyme II (hACE2) and became more infectious. Using this SEM pseudovirus, we examined the infectivity and antigenic properties of the natural SARS-CoV-2 variants. We showed that some variants have higher infectivity than the original virus and that some render the neutralizing plasma with lower potency. These studies thus revealed possible mechanisms of the dissemination advantage of these variants. Hence, the SEM pseudovirion provides a useful tool to evaluate the viral infectivity and capability of convalescent sera in neutralizing specific SARS-CoV-2 S dominant variants.


Assuntos
Anticorpos Antivirais/metabolismo , COVID-19/imunologia , Proteínas do Envelope de Coronavírus/metabolismo , SARS-CoV-2/patogenicidade , Proteínas da Matriz Viral/metabolismo , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , Anticorpos Antivirais/imunologia , COVID-19/sangue , COVID-19/virologia , Linhagem Celular , Proteínas do Envelope de Coronavírus/genética , Proteínas do Envelope de Coronavírus/imunologia , Proteínas do Envelope de Coronavírus/ultraestrutura , Cricetinae , Humanos , Microscopia Eletrônica de Transmissão , Mutação , Testes de Neutralização , SARS-CoV-2/genética , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/imunologia , Proteínas da Matriz Viral/ultraestrutura , Vírion/genética , Vírion/imunologia , Vírion/metabolismo , Vírion/ultraestrutura
9.
Zhongguo Zhong Yao Za Zhi ; 46(14): 3465-3477, 2021 Jul.
Artigo em Chinês | MEDLINE | ID: mdl-34402268

RESUMO

High fat diet induced hyperlipidemia hamster model was used to explore the anti-hyperlipidemia effect of water extract of Moringa oleifera leaves( WEMOL). On this basis,the possible action mechanism was predicted by network pharmacology. Golden hamsters were randomly divided into normal diet group( NFD),high-fat diet group( HFD),simvastatin group,high dose group of WEMOL( HIWEMOL) and low dose group of WEMOL( LOWEMOL). The model was administered simultaneously for 66 days,during which the body weight changes of hamsters were recorded. At the end of the experiment,serum lipid level and serum transaminase level of golden hamsters in each group were detected,and the pathological changes of liver were observed by hematoxylin-eosin( HE) staining. The results showed that WEMOL could significantly decrease the serum total cholesterol( TC),total triglyceride( TG),low density lipoprotein cholesterol( LDL-c) levels,and reduce the lipid deposition in liver tissue,thus improving the hyperlipidemia of golden hamsters. According to the prediction of network pharmacology,219 targets of potential active components of M.oleifera leaves and 185 targets of water-soluble potential active components of M. oleifera leaves for the treatment of hyperlipidemia were obtained separately. The MCODE analysis was performed on the PPI network of 219 targets and 185 targets obtained above and got five and four clusters respectively. The signaling pathway analysis of clusters showed that among the common pathways,nonalcoholic fatty liver,insulin resistance,MAPK signaling pathway,estrogen signaling pathway,cell apoptosis and HIF-1 signaling pathway were associated with hyperlipidemia. In addition,the potential active components of M. oleifera leaves could also inhibit the metabolic inflammation of hyperlipidemia by modulating complement and coagulation cascades signaling pathway,and GSK3 B,F2,AKT1,RELA,SERPINE1 might be the key targets. The water-soluble potential active components of M. oliefera leaves could modulate lipid metabolism by modulating AMPK signaling pathway and JAK-STAT signaling pathway,with PIK3 CB,PIK3 CA,CASP3,AKT1 and BCL2 as the key targets. These results suggested that WEMOL had anti hyperlipidemia effect,and its mechanism might be related to the protein expression regulation of lipid metabolism,nonalcoholic fatty liver disease and atherosclerosis related signaling pathways.


Assuntos
Hiperlipidemias , Moringa oleifera , Animais , Cricetinae , Dieta Hiperlipídica , Quinase 3 da Glicogênio Sintase , Hiperlipidemias/tratamento farmacológico , Fígado , Folhas de Planta
10.
J Gen Virol ; 102(8)2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34424160

RESUMO

Senecavirus A (SVA) is a picornavirus that circulates in swine populations worldwide causing vesicular disease (VD) in affected animals. Here we developed a reverse genetics system for SVA based on the well-characterized wild-type SVA strain SD15-26 (wt SVA SD15-26). The full-length cDNA genome of SVA was cloned into a plasmid under a T7 RNA polymerase promoter. Following in vitro transcription, the genomic viral RNA was transfected into BHK-21 cells and rescue of infectious virus (rSVA SD15-26) was shown by inoculation of highly susceptible H1299 cells. In vitro characterization of the rSVA SD15-26 showed similar replication properties and protein expression levels as the wt SVA SD15-26. A pathogenesis study was conducted in 15-week-old finishing pigs to evaluate the pathogenicity and infection dynamics of the rSVA SD15-26 virus in comparison to the wt SVA SD15-26. Animals from both rSVA- and wt SVA SD15-26-inoculated groups presented characteristic SVA clinical signs (lethargy and lameness) followed by the development of vesicular lesions on the snout and/or feet. The clinical outcome of infection, including disease onset, severity and duration was similar in rSVA- and the wt SVA SD15-26-inoculated animals. All animals inoculated with rSVA or with wt SVA SD15-26 presented a short-term viremia, and animals from both groups shed similar amounts of virus in oral and nasal secretion, and faeces. Our data demonstrates that the rSVA SD5-26 clone is fully virulent and pathogenic in pigs, presenting comparable pathogenesis and infection dynamics to the wt SVA SD15-26 strain. The infectious clone generated here is a useful platform to study virulence determinants of SVA, and to dissect other aspects of SVA infection biology, pathogenesis and persistence.


Assuntos
Infecções por Picornaviridae , Picornaviridae/patogenicidade , Doenças dos Suínos/virologia , Animais , Linhagem Celular , Cricetinae , Humanos , Infecções por Picornaviridae/veterinária , Infecções por Picornaviridae/virologia , Suínos , Viremia/virologia , Virulência
11.
Commun Biol ; 4(1): 959, 2021 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-34381146

RESUMO

The association between kidney stone disease and renal fibrosis has been widely explored in recent years but its underlying mechanisms remain far from complete understanding. Using label-free quantitative proteomics (nanoLC-ESI-LTQ-Orbitrap MS/MS), this study identified 23 significantly altered secreted proteins from calcium oxalate monohydrate (COM)-exposed macrophages (COM-MP) compared with control macrophages (Ctrl-MP) secretome. Functional annotation and protein-protein interactions network analysis revealed that these altered secreted proteins were involved mainly in inflammatory response and fibroblast activation. BHK-21 renal fibroblasts treated with COM-MP secretome had more spindle-shaped morphology with greater spindle index. Immunofluorescence study and gelatin zymography revealed increased levels of fibroblast activation markers (α-smooth muscle actin and F-actin) and fibrotic factors (fibronectin and matrix metalloproteinase-9 and -2) in the COM-MP secretome-treated fibroblasts. Our findings indicate that proteins secreted from macrophages exposed to COM crystals induce renal fibroblast activation and may play important roles in renal fibrogenesis in kidney stone disease.


Assuntos
Oxalato de Cálcio/metabolismo , Fibroblastos/metabolismo , Rim/metabolismo , Macrófagos/metabolismo , Animais , Oxalato de Cálcio/química , Cricetinae , Humanos , Mapas de Interação de Proteínas , Células U937
12.
PLoS Pathog ; 17(8): e1009427, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34370799

RESUMO

Impaired type I interferons (IFNs) production or signaling have been associated with severe COVID-19, further promoting the evaluation of recombinant type I IFNs as therapeutics against SARS-CoV-2 infection. In the Syrian hamster model, we show that intranasal administration of IFN-α starting one day pre-infection or one day post-infection limited weight loss and decreased viral lung titers. By contrast, intranasal administration of IFN-α starting at the onset of symptoms three days post-infection had no impact on the clinical course of SARS-CoV-2 infection. Our results provide evidence that early type I IFN treatment is beneficial, while late interventions are ineffective, although not associated with signs of enhanced disease.


Assuntos
Antivirais/administração & dosagem , COVID-19/tratamento farmacológico , Interferon Tipo I/administração & dosagem , Administração Intranasal , Animais , Chlorocebus aethiops , Cricetinae , Modelos Animais de Doenças , Mesocricetus , SARS-CoV-2
13.
Front Immunol ; 12: 719077, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34394127

RESUMO

The COVID-19 pandemic caused by the coronavirus SARS-CoV-2 is a major global public threat. Currently, a worldwide effort has been mounted to generate billions of effective SARS-CoV-2 vaccine doses to immunize the world's population at record speeds. However, there is still a demand for alternative effective vaccines that rapidly confer long-term protection and rely upon cost-effective, easily scaled-up manufacturing. Here, we present a Sindbis alphavirus vector (SV), transiently expressing the SARS-CoV-2 spike protein (SV.Spike), combined with the OX40 immunostimulatory antibody (αOX40) as a novel, highly effective vaccine approach. We show that SV.Spike plus αOX40 elicits long-lasting neutralizing antibodies and a vigorous T-cell response in mice. Protein binding, immunohistochemical, and cellular infection assays all show that vaccinated mice sera inhibits spike functions. Immunophenotyping, RNA Seq transcriptome profiles, and metabolic analysis indicate a reprogramming of T cells in vaccinated mice. Activated T cells were found to mobilize to lung tissue. Most importantly, SV.Spike plus αOX40 provided robust immune protection against infection with authentic coronavirus in transgenic mice expressing the human ACE2 receptor (hACE2-Tg). Finally, our immunization strategy induced strong effector memory response, potentiating protective immunity against re-exposure to SARS-CoV-2 spike protein. Our results show the potential of a new Sindbis virus-based vaccine platform to counteract waning immune response, which can be used as a new candidate to combat SARS-CoV-2. Given the T-cell responses elicited, our vaccine is likely to be effective against variants that are proving challenging, as well as serve as a platform to develop a broader spectrum pancoronavirus vaccine. Similarly, the vaccine approach is likely to be applicable to other pathogens.


Assuntos
Antígenos de Diferenciação/imunologia , Vacinas contra COVID-19/imunologia , COVID-19/prevenção & controle , SARS-CoV-2/imunologia , Vírus Sindbis/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , COVID-19/imunologia , Cricetinae , Feminino , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Vírus Sindbis/genética , Linfócitos T/imunologia , Vacinação
14.
FEMS Microbiol Lett ; 368(16)2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34459482

RESUMO

Limited research exists on the potential for leather to act as a fomite of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or endemic coronaviruses including human coronavirus (HCoV) OC43; this is important for settings such as the shoe manufacturing industry. Antiviral coating of leather hides could limit such risks. This study aimed to investigate the stability and transfer of HCoVOC43 on different leathers, as a surrogate for SARS-CoV-2, and assess the antiviral efficacy of a silver-based leather coating. The stability of HCoV-OC43 (6.6 log10) on patent, full-grain calf, corrected grain finished and nubuck leathers (silver additive-coated and uncoated) was measured by titration on BHK-21 cells. Transfer from leather to cardboard and stainless steel was determined. HCoV-OC43 was detectable for 6 h on patent, 24 h on finished leather and 48 h on calf leather; no infectious virus was recovered from nubuck. HCoV-OC43 transferred from patent, finished and calf leathers onto cardboard and stainless steel up to 2 h post-inoculation (≤3.1-5.5 log10), suggesting that leathers could act as fomites. Silver additive-coated calf and finished leathers were antiviral against HCoV-OC43, with no infectious virus recovered after 2 h and limited transfer to other surfaces. The silver additive could reduce potential indirect transmission of HCoV-OC43 from leather.


Assuntos
Infecções por Coronavirus/transmissão , Coronavirus Humano OC43/isolamento & purificação , Fômites/virologia , Animais , Antivirais/farmacologia , COVID-19/transmissão , Linhagem Celular , Coronavirus Humano OC43/efeitos dos fármacos , Cricetinae , Transmissão de Doença Infecciosa/prevenção & controle , Fômites/classificação , Humanos , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/isolamento & purificação , Prata/farmacologia
15.
Emerg Microbes Infect ; 10(1): 1790-1806, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34427172

RESUMO

The unprecedented in recent history global COVID-19 pandemic urged the implementation of all existing vaccine platforms to ensure the availability of the vaccines against COVID-19 to every country in the world. Despite the multitude of high-quality papers describing clinical trials of different vaccine products, basic detailed data on general toxicity, reproductive toxicity, immunogenicity, protective efficacy and durability of immune response in animal models are scarce. Here, we developed a ß-propiolactone-inactivated whole virion vaccine CoviVac and assessed its safety, protective efficacy, immunogenicity and stability of the immune response in rodents and non-human primates. The vaccine showed no signs of acute/chronic, reproductive, embryo- and fetotoxicity, or teratogenic effects, as well as no allergenic properties in studied animal species. The vaccine induced stable and robust humoral immune response both in form of specific anti-SARS-CoV-2 IgG and NAbs in mice, Syrian hamsters, and common marmosets. The NAb levels did not decrease significantly over the course of one year. The course of two immunizations protected Syrian hamsters from severe pneumonia upon intranasal challenge with the live virus. Robustness of the vaccine manufacturing process was demonstrated as well. These data encouraged further evaluation of CoviVac in clinical trials.


Assuntos
Vacinas contra COVID-19/imunologia , COVID-19/prevenção & controle , Imunidade Humoral , SARS-CoV-2/imunologia , Vacinas de Produtos Inativados/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , COVID-19/imunologia , COVID-19/virologia , Vacinas contra COVID-19/administração & dosagem , Vacinas contra COVID-19/efeitos adversos , Callithrix , Cricetinae , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Feminino , Cobaias , Humanos , Imunogenicidade da Vacina , Imunoglobulina G/imunologia , Masculino , Mesocricetus , Camundongos , Camundongos Endogâmicos BALB C , Ratos , Ratos Wistar , SARS-CoV-2/genética , Fatores de Tempo , Vacinas de Produtos Inativados/administração & dosagem , Vacinas de Produtos Inativados/efeitos adversos
16.
Int J Mol Sci ; 22(16)2021 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-34445287

RESUMO

Bovine herpesvirus 1 (BoHV-1) is a promising oncolytic virus with broad antitumor spectrum; however, its oncolytic effects on human lung adenocarcinoma in vivo have not been reported. In this study, we report that BoHV-1 can be used as an oncolytic virus for human lung adenocarcinoma, and elucidate the underlying mechanism of how BoHV-1 suppresses tumor cell proliferation and growth. First, we examined the oncolytic activities of BoHV-1 in human lung adenocarcinoma A549 cells. BoHV-1 infection reduced the protein levels of histone deacetylases (HDACs), including HDAC1-4 that are promising anti-tumor drug targets. Furthermore, the HDAC inhibitor Trichostatin A (TSA) promoted BoHV-1 infection and exacerbated DNA damage and cytopathology, suggesting a synergy between BoHV-1 and TSA. In the A549 tumor xenograft mouse model, we, for the first time, showed that BoHV-1 can infect tumor and suppressed tumor growth with a similar high efficacy as the treatment of TSA, and HDACs have potential effects on the virus replication. Taken together, our study demonstrates that BoHV-1 has oncolytic effects against human lung adenocarcinoma in vivo.


Assuntos
Adenocarcinoma de Pulmão/patologia , Herpesvirus Bovino 1/fisiologia , Neoplasias Pulmonares/patologia , Células A549 , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/terapia , Adenocarcinoma de Pulmão/virologia , Animais , Proliferação de Células/genética , Células Cultivadas , Cricetinae , Dano ao DNA , Feminino , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/terapia , Neoplasias Pulmonares/virologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Terapia Viral Oncolítica/métodos , Vírus Oncolíticos/fisiologia , Ensaios Antitumorais Modelo de Xenoenxerto
17.
Nat Microbiol ; 6(9): 1188-1198, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34400835

RESUMO

SARS-CoV-2 variants of interest and concern will continue to emerge for the duration of the COVID-19 pandemic. To map mutations in the receptor-binding domain (RBD) of the spike protein that affect binding to angiotensin-converting enzyme 2 (ACE2), the receptor for SARS-CoV-2, we applied in vitro evolution to affinity-mature the RBD. Multiple rounds of random mutagenic libraries of the RBD were sorted against decreasing concentrations of ACE2, resulting in the selection of higher affinity RBD binders. We found that mutations present in more transmissible viruses (S477N, E484K and N501Y) were preferentially selected in our high-throughput screen. Evolved RBD mutants include prominently the amino acid substitutions found in the RBDs of B.1.620, B.1.1.7 (Alpha), B1.351 (Beta) and P.1 (Gamma) variants. Moreover, the incidence of RBD mutations in the population as presented in the GISAID database (April 2021) is positively correlated with increased binding affinity to ACE2. Further in vitro evolution increased binding by 1,000-fold and identified mutations that may be more infectious if they evolve in the circulating viral population, for example, Q498R is epistatic to N501Y. We show that our high-affinity variant RBD-62 can be used as a drug to inhibit infection with SARS-CoV-2 and variants Alpha, Beta and Gamma in vitro. In a model of SARS-CoV-2 challenge in hamster, RBD-62 significantly reduced clinical disease when administered before or after infection. A 2.9 Å cryo-electron microscopy structure of the high-affinity complex of RBD-62 and ACE2, including all rapidly spreading mutations, provides a structural basis for future drug and vaccine development and for in silico evaluation of known antibodies.


Assuntos
Enzima de Conversão de Angiotensina 2/metabolismo , Antivirais/administração & dosagem , COVID-19/virologia , SARS-CoV-2/efeitos dos fármacos , Glicoproteína da Espícula de Coronavírus/metabolismo , Enzima de Conversão de Angiotensina 2/química , Enzima de Conversão de Angiotensina 2/genética , Animais , Antivirais/química , COVID-19/tratamento farmacológico , COVID-19/genética , COVID-19/metabolismo , Cricetinae , Desenho de Fármacos , Evolução Molecular , Feminino , Humanos , Masculino , Mesocricetus , Simulação de Dinâmica Molecular , Mutação , Ligação Proteica/efeitos dos fármacos , Domínios Proteicos , Receptores Virais/genética , Receptores Virais/metabolismo , SARS-CoV-2/química , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Internalização do Vírus/efeitos dos fármacos
18.
Vaccine ; 39(36): 5153-5161, 2021 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-34362602

RESUMO

Members of the Developing Countries Vaccine Manufacturers' Network (DCVMN) have been actively engaged in the development of COVID-19 vaccine candidates. According to the WHO COVID-19 vaccine landscape updated on 29 December 2020, 18 member manufacturers had vaccines in preclinical or clinical trials, including three members with candidates in Phase III trials. Once successful candidates have been identified there will be a need for large scale vaccine manufacturing and supply, in which DCVMN member manufacturers can play a key role. In an internal survey in 2019, DCVMN members reported the capability to supply over 3.5 billion vaccine doses annually, and the provision of over 50 distinct vaccines to 170 countries. To describe the capabilities of DCVMN member manufacturers more precisely, a 121-question survey was circulated to 41 Network members. The survey assessed the manufacturers' capabilities in utilizing various technology platforms, cell cultures and filling technologies, in addition to their capacities for manufacturing drug products. The survey also evaluated manufacturers' preparedness to dedicate existing capacities to COVID-19 vaccine production. Results revealed that sampled manufacturers have strong capabilities for manufacturing vaccines based on recombinant technologies, particularly with mammalian cells, and microbial and yeast expression systems. Capabilities in utilizing cell cultures were distributed across multiple cell types, however manufacturing capacities with Vero and CHO cells were prominent. Formulating and filling findings illustrated further large-scale capabilities of Network members. Sampled manufacturers reported that over 50% of their capacity for vaccine manufacturing could be dedicated to COVID-19 vaccine production.


Assuntos
COVID-19 , Vacinas , Animais , Vacinas contra COVID-19 , Cricetinae , Cricetulus , Países em Desenvolvimento , Humanos , Imunização , SARS-CoV-2
19.
Environ Pollut ; 285: 117527, 2021 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-34380225

RESUMO

2-Ethylhexyl diphenyl phosphate (EHDPP) is a common flame retardant and environmental pollutant, exposing humans with endocrinal disrupting potentials. Its mutagenicity, especially following metabolism, remains unclear. In this study, molecular docking analysis indicated that EHDPP was a potential substrate for several human CYP enzymes except for CYP1A1. Among V79-derived cell lines genetically engineered for the expression of each CYP, EHDPP (6 h exposure/18 h recovery) did not induce micronuclei in the V79 or V79-derived cells expressing human CYP1A1, however, it was positive in V79-derived cell lines expressing human CYP2E1, 3A4, and 2B6. In a human hepatoma (HepG2) cell line, EHDPP (48 h exposure) moderately induced micronuclei, which was blocked by 1-aminobenzotriazole (ABT, 60 µM, inhibitor of CYPs); pretreating HepG2 cells with bisphenol AF, another organic pollutant as inducer of CYPs (0.1 µM for 16 h), significantly potentiated micronuclei formation by EHDPP, threshold being decreased from 10 to 1.25 µM. This effect was blocked by ABT, drastically reduced by ketoconazole (inhibiting CYP3A expression/activity), and moderately inhibited by trans-1,2-dichloroethylene (selective CYP2E1 inhibitor). Immunofluorescent centromere protein B staining indicated that EHDPP-induced micronuclei in V79-derived cell lines expressing human CYP2E1 and 3A4 were predominantly centromere-negative, and that in HepG2 cells pretreated with bisphenol AF (for inducing multiple CYPs) were purely centromere-negative. In bisphenol AF-pretreated HepG2 cells EHDPP potently induced DNA breaks, as indicated by the comet assay and Western blot analysis of γ-H2AX. In conclusion, our study suggests that EHDPP is potently clastogenic, following activation by several human CYP enzymes, CYP3A4 being a major one.


Assuntos
Retardadores de Chama , Mutagênicos , Animais , Compostos de Bifenilo , Linhagem Celular , Cricetinae , Cricetulus , Retardadores de Chama/toxicidade , Humanos , Simulação de Acoplamento Molecular , Fosfatos
20.
Artigo em Inglês | MEDLINE | ID: mdl-34378764

RESUMO

Chagas disease (CD) has been changing from an endemic Latino-American disease to a condition found outside endemic regions, due to migratory movements. Although often subclinical, its acute phase can be lethal. This study aimed to assess survival during the acute phase of CD and its relationship with ventricular function in an experimental model. To this end, 30 Syrian hamsters were inoculated with Trypanosoma cruzi (IG) and other 15 animals received saline solution (CG). Groups were monitored daily and submitted to echocardiography in two moments: before the challenge and 15 days post-infection. Left ventricular ejection fraction (LVEF) and global longitudinal myocardial strain (GLS) of the LV were measured. The IG was divided into groups of animals with and without clinical signs of disease. ANOVA for mixed models was used to compare ventricular function parameters. Survival analysis was studied using Kaplan-Meier curves and the log-rank test. The follow-up lasted 60 days. LVEF in IG was reduced through time (53.80 to 43.55%) compared to CG (57.86 to 59.73%) (p=0.002). There was also a reduction of GLS (-18.97% to -12.44%) in the IG compared to CG (p=0.012). Twelve animals from IG died compared to one animal from CG. Eleven out of the 12 animals from the IG group died before presenting with clinical signs of infection. Survival was reduced in the IG compared to CG over time (p=0.02). The reduced survival during the acute phase of this experimental model of Chagas disease was related to the significant reduction of LV function. The mortality rate in the IG was higher in the group presenting with clinical signs of infection.


Assuntos
Doença de Chagas , Disfunção Ventricular Esquerda , Animais , Cricetinae , Modelos Animais , Estudos Prospectivos , Volume Sistólico , Função Ventricular Esquerda
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