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2.
Signal Transduct Target Ther ; 6(1): 438, 2021 12 24.
Artigo em Inglês | MEDLINE | ID: mdl-34952914

RESUMO

Messenger RNA (mRNA) vaccine technology has shown its power in preventing the ongoing COVID-19 pandemic. Two mRNA vaccines targeting the full-length S protein of SARS-CoV-2 have been authorized for emergency use. Recently, we have developed a lipid nanoparticle-encapsulated mRNA (mRNA-LNP) encoding the receptor-binding domain (RBD) of SARS-CoV-2 (termed ARCoV), which confers complete protection in mouse model. Herein, we further characterized the protection efficacy of ARCoV in nonhuman primates and the long-term stability under normal refrigerator temperature. Intramuscular immunization of two doses of ARCoV elicited robust neutralizing antibodies as well as cellular response against SARS-CoV-2 in cynomolgus macaques. More importantly, ARCoV vaccination in macaques significantly protected animals from acute lung lesions caused by SARS-CoV-2, and viral replication in lungs and secretion in nasal swabs were completely cleared in all animals immunized with low or high doses of ARCoV. No evidence of antibody-dependent enhancement of infection was observed throughout the study. Finally, extensive stability assays showed that ARCoV can be stored at 2-8 °C for at least 6 months without decrease of immunogenicity. All these promising results strongly support the ongoing clinical trial.


Assuntos
Vacinas contra COVID-19/farmacologia , COVID-19/imunologia , Imunogenicidade da Vacina , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , /farmacologia , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , COVID-19/prevenção & controle , Vacinas contra COVID-19/imunologia , Chlorocebus aethiops , Humanos , Macaca fascicularis , Células Vero , /imunologia
5.
J Craniofac Surg ; 2021 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-34789671

RESUMO

ABSTRACT: In this report, the authors describe a case of the acute anterior disc displacement without reduction treated by manipulative reduction combined with the disc-condyle repositioning splint to improve the limited mouth opening and relieve the pain, including diagnostic images and treatment performed.

6.
Signal Transduct Target Ther ; 6(1): 389, 2021 11 10.
Artigo em Inglês | MEDLINE | ID: mdl-34759261

RESUMO

SARS-CoV-2 and SARS-CoV are genetically related coronavirus and share the same cellular receptor ACE2. By replacing the VSV glycoprotein with the spikes (S) of SARS-CoV-2 and SARS-CoV, we generated two replication-competent recombinant viruses, rVSV-SARS-CoV-2 and rVSV-SARS-CoV. Using wild-type and human ACE2 (hACE2) knock-in mouse models, we found a single dose of rVSV-SARS-CoV could elicit strong humoral immune response via both intranasal (i.n.) and intramuscular (i.m.) routes. Despite the high genetic similarity between SARS-CoV-2 and SARS-CoV, no obvious cross-neutralizing activity was observed in the immunized mice sera. In macaques, neutralizing antibody (NAb) titers induced by one i.n. dose of rVSV-SARS-CoV-2 were eight-fold higher than those by a single i.m. dose. Thus, our data indicates that rVSV-SARS-CoV-2 might be suitable for i.n. administration instead of the traditional i.m. immunization in human. Because rVSV-SARS-CoV elicited significantly stronger NAb responses than rVSV-SARS-CoV-2 in a route-independent manner, we generated a chimeric antigen by replacing the receptor binding domain (RBD) of SARS-CoV S with that from the SARS-CoV-2. rVSV expressing the chimera (rVSV-SARS-CoV/2-RBD) induced significantly increased NAbs against SARS-CoV-2 in mice and macaques than rVSV-SARS-CoV-2, with a safe Th1-biased response. Serum immunized with rVSV-SARS-CoV/2-RBD showed no cross-reactivity with SARS-CoV. hACE2 mice receiving a single i.m. dose of either rVSV-SARS-CoV-2 or rVSV-SARS-CoV/2-RBD were fully protected against SARS-CoV-2 challenge without obvious lesions in the lungs. Our results suggest that transplantation of SARS-CoV-2 RBD into the S protein of SARS-CoV might be a promising antigen design for COVID-19 vaccines.


Assuntos
Vacinas contra COVID-19/imunologia , COVID-19/prevenção & controle , Glicoproteína da Espícula de Coronavírus/imunologia , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Técnicas de Introdução de Genes , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Testes de Neutralização , Proteínas Recombinantes de Fusão/imunologia , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus/genética
7.
Natl Sci Rev ; 8(3): nwaa297, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-34676096

RESUMO

Receptor recognition and subsequent membrane fusion are essential for the establishment of successful infection by SARS-CoV-2. Halting these steps can cure COVID-19. Here we have identified and characterized a potent human monoclonal antibody, HB27, that blocks SARS-CoV-2 attachment to its cellular receptor at sub-nM concentrations. Remarkably, HB27 can also prevent SARS-CoV-2 membrane fusion. Consequently, a single dose of HB27 conferred effective protection against SARS-CoV-2 in two established mouse models. Rhesus macaques showed no obvious adverse events when administrated with 10 times the effective dose of HB27. Cryo-EM studies on complex of SARS-CoV-2 trimeric S with HB27 Fab reveal that three Fab fragments work synergistically to occlude SARS-CoV-2 from binding to the ACE2 receptor. Binding of the antibody also restrains any further conformational changes of the receptor binding domain, possibly interfering with progression from the prefusion to the postfusion stage. These results suggest that HB27 is a promising candidate for immuno-therapies against COVID-19.

8.
Natl Sci Rev ; 8(8): nwab053, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34676098

RESUMO

Mutations and transient conformational movements of the receptor binding domain (RBD) that make neutralizing epitopes momentarily unavailable present immune escape routes for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To mitigate viral escape, we developed a cocktail of neutralizing antibodies (NAbs) targeting epitopes located on different domains of spike (S) protein. Screening of a library of monoclonal antibodies generated from peripheral blood mononuclear cells of COVID-19 convalescent patients yielded potent NAbs, targeting the N-terminal domain (NTD) and RBD domain of S, effective at nM concentrations. Remarkably, a combination of RBD-targeting NAbs and NTD-binding NAbs, FC05, enhanced the neutralization potency in cell-based assays and an animal model. Results of competitive surface plasmon resonance assays and cryo-electron microscopy (cryo-EM) structures of antigen-binding fragments bound to S unveil determinants of immunogenicity. Combinations of immunogens, identified in the NTD and RBD of S, when immunized in rabbits and macaques, elicited potent protective immune responses against SARS-CoV-2. More importantly, two immunizations of this combination of NTD and RBD immunogens provided complete protection in macaques against a SARS-CoV-2 challenge, without observable antibody-dependent enhancement of infection. These results provide a proof of concept for neutralization-based immunogen design targeting SARS-CoV-2 NTD and RBD.

9.
Nat Commun ; 12(1): 5654, 2021 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-34580297

RESUMO

There is an urgent need for animal models to study SARS-CoV-2 pathogenicity. Here, we generate and characterize a novel mouse-adapted SARS-CoV-2 strain, MASCp36, that causes severe respiratory symptoms, and mortality. Our model exhibits age- and gender-related mortality akin to severe COVID-19. Deep sequencing identified three amino acid substitutions, N501Y, Q493H, and K417N, at the receptor binding domain (RBD) of MASCp36, during in vivo passaging. All three RBD mutations significantly enhance binding affinity to its endogenous receptor, ACE2. Cryo-electron microscopy analysis of human ACE2 (hACE2), or mouse ACE2 (mACE2), in complex with the RBD of MASCp36, at 3.1 to 3.7 Å resolution, reveals the molecular basis for the receptor-binding switch. N501Y and Q493H enhance the binding affinity to hACE2, whereas triple mutations at N501Y/Q493H/K417N decrease affinity and reduce infectivity of MASCp36. Our study provides a platform for studying SARS-CoV-2 pathogenesis, and unveils the molecular mechanism for its rapid adaptation and evolution.


Assuntos
COVID-19/diagnóstico , SARS-CoV-2/patogenicidade , Glicoproteína da Espícula de Coronavírus/metabolismo , Substituição de Aminoácidos , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , Sítios de Ligação/genética , COVID-19/mortalidade , COVID-19/virologia , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Ligação Proteica/genética , Domínios Proteicos/genética , SARS-CoV-2/genética , Índice de Gravidade de Doença , Glicoproteína da Espícula de Coronavírus/genética
10.
Emerg Microbes Infect ; 10(1): 1739-1750, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34379047

RESUMO

Yellow fever virus (YFV) is a re-emerging flavivirus, which can lead to severe clinical manifestations and high mortality, with no specific antiviral therapies available. The live-attenuated yellow fever vaccine 17D (YF17D) has been widely used for over eighty years. However, the emergence of yellow fever vaccine-associated viscerotropic disease (YFL-AVD) and yellow fever vaccine-associated neurotropic disease (YFL-AND) raised non-negligible concerns. Additionally, the attenuation mechanism of YF17D is still unclear. Thus, the development of convenient models is crucial to understand the mechanisms behind YF17D attenuation and its adverse effects. In this work, we generated a reporter YF17D expressing nano-luciferase (NLuc). In vitro and in vivo characterization demonstrated that the NLuc-YF17D shared similar biological properties with its parental strain and the NLuc activity can reflect viral infectivity reliably. Combined with in vivo bioluminescence imaging, a series of mice models of YF17D infection was established, which will be useful for the evaluation of antiviral medicines and novel vaccine candidates. Especially, we demonstrated that intraperitoneally (i.p.) infection of NLuc-YF17D in type I interferon receptor-deficient mice A129 resulted in outcomes resembling YEL-AVD and YEL-AND, evidenced by viral replication in multiple organs and invasion of the central neuronal system. Finally, in vitro and in vivo assays based on this reporter virus were established to evaluate the antiviral activities of validated antiviral agents. In conclusion, the bioluminescent reporter virus described herein provides a powerful platform to study YF17D attenuation and vaccine-associated diseases as well as to develop novel countermeasures against YFV.

11.
J Craniofac Surg ; 2021 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-34387262

RESUMO

OBJECTIVE: This study aims to investigate the clinical effects of the combination of rhytidectomy and temporomandibular joint (TMJ) disc repositioning surgery in internal derangement (ID) stage IV-V and facial aging patients. METHODS: Eighteen facial aging with bilateral ID IV-V patients were enrolled in this study. All of them had undergone temporomandibular disc repositioning surgery and rhytidectomy by the same surgeon (Yao Min Zhu). Pre-/post-surgical clinical manifestations, facial photography, radiographic data were recorded and analyzed, as well as doctor, patient, third-party evaluation of postsurgical facial appearance satisfaction. RESULTS: The average age of 18 female patients was 52.9. The average of presurgical visual analog pain scale score was 5.94, ranged from 4 to 8. After 6 months, the average of postsurgical visual analog pain scale score was 0.28, ranged from 0 to 1 (P > 0.05). The average maximal mouth opening of presurgical and postsurgical was 2.19 and 3.29 cm, ranged from 1.2 to 2.8 cm and 3.0 to 3.5 cm, respectively (P < 0.05). Postoperative magnetic resonance imaging showed the location of the bilateral TMJ discs directly above the mandibular condyle. The satisfaction rate of doctors, patients and third-party with facial appearance was 95% to 98%, 96% to 99% and 96% to 99%, respectively, with an average of 95.72%, 98.11%, and 97.50%. CONCLUSIONS: For patients with bilateral ID IV-V and facial aging, the combination of disc repositioning surgery and rhytidectomy is a very feasible procedure to treat TMJ disorders and improve patients' facial appearance and satisfaction.

12.
Virol Sin ; 2021 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-34403037

RESUMO

The sudden emergence of severe acute respiratory syndrome coronavirus (SARS-CoV) has caused global panic in 2003, and the risk of SARS-CoV outbreak still exists. However, no specific antiviral drug or vaccine is available; thus, the development of therapeutic antibodies against SARS-CoV is needed. In this study, a nanobody phage-displayed library was constructed from peripheral blood mononuclear cells of alpacas immunized with the recombinant receptor-binding domain (RBD) of SARS-CoV. Four positive clones were selected after four rounds of bio-panning and subjected to recombinant expression in E. coli. Further biological identification demonstrated that one of the nanobodies, S14, showed high affinity to SARS-CoV RBD and potent neutralization activity at the picomole level against SARS-CoV pseudovirus. A competitive inhibition assay showed that S14 blocked the binding of SARS-CoV RBD to either soluble or cell-expressed angiotensin-converting enzyme 2 (ACE2). In summary, we developed a novel nanobody targeting SARS-CoV RBD, which might be useful for the development of therapeutics against SARS.

13.
Front Cell Infect Microbiol ; 11: 687338, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34249779

RESUMO

Sexual transmission of Zika Virus (ZIKV) elevates the risk of its dissemination in the female reproductive tract and causes a serious threat to the fetus. However, the available animal models are not appropriate to investigate sexual transmission, dynamics of ZIKV infection, replication, and shedding. The use of tree shrew as a small animal model of ZIKV vaginal infection was assessed in this study. A total of 23 sexually mature female tree shrews were infected with ZIKV GZ01 via the intravaginal route. There was no significant difference in change of body weight, and the temperature between ZIKV infected and control animals. Viral RNA loads were detected in blood, saliva, urine, and vaginal douching. ZIKV RNA was readily detected in vaginal lavage of 22 animals (95.65%, 22/23) at 1 dpi, and viral load ranged from 104.46 to 107.35 copies/ml, and the peak of viral load appeared at 1 dpi. The expression of key inflammatory genes, such as IL6, 8, CCL5, TNF-a, and CXCL9, was increased in the spleen of ZIKV infected animals. In the current study, female tree shrews have been successfully infected with ZIKV through the vaginal route for the first time. Interestingly, at first, ZIKV replicates at the local site of infection and then spreads throughout the host body to develop a robust systemic infection and mounted a protective immune response. This small animal model is not only valuable for exploring ZIKV sexual transmission and may also help to explain the cause of debilitating manifestations of the fetus in vivo.


Assuntos
Infecção por Zika virus , Zika virus , Animais , Modelos Animais de Doenças , Feminino , Tupaia , Tupaiidae , Vagina
14.
Cell Discov ; 7(1): 49, 2021 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-34230457

RESUMO

SARS-CoV-2 infection causes a wide spectrum of clinical manifestations in humans, and olfactory dysfunction is one of the most predictive and common symptoms in COVID-19 patients. However, the underlying mechanism by which SARS-CoV-2 infection leads to olfactory disorders remains elusive. Herein, we demonstrate that intranasal inoculation with SARS-CoV-2 induces robust viral replication in the olfactory epithelium (OE), not the olfactory bulb (OB), resulting in transient olfactory dysfunction in humanized ACE2 (hACE2) mice. The sustentacular cells and Bowman's gland cells in the OE were identified as the major target cells of SARS-CoV-2 before invasion into olfactory sensory neurons (OSNs). Remarkably, SARS-CoV-2 infection triggers massive cell death and immune cell infiltration and directly impairs the uniformity of the OE structure. Combined transcriptomic and quantitative proteomic analyses revealed the induction of antiviral and inflammatory responses, as well as the downregulation of olfactory receptor (OR) genes in the OE from the infected animals. Overall, our mouse model recapitulates olfactory dysfunction in COVID-19 patients and provides critical clues for understanding the physiological basis for extrapulmonary manifestations of COVID-19.

16.
Commun Biol ; 4(1): 500, 2021 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-33893388

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the ongoing COVID-19 pandemic, which has resulted in more than two million deaths at 2021 February . There is currently no approved therapeutics for treating COVID-19. The SARS-CoV-2 Spike protein is considered a key therapeutic target by many researchers. Here we describe the identification of several monoclonal antibodies that target SARS-CoV-2 Spike protein. One human antibody, CA521FALA, demonstrated neutralization potential by immunizing human antibody transgenic mice. CA521FALA showed potent SARS-CoV-2-specific neutralization activity against SARS-CoV-2 pseudovirus and authentic SARS-CoV-2 infection in vitro. CA521FALA also demonstrated having a long half-life of 9.5 days in mice and 9.3 days in rhesus monkeys. CA521FALA inhibited SARS-CoV-2 infection in SARS-CoV-2 susceptible mice at a therapeutic setting with virus titer of the lung reduced by 4.5 logs. Structural analysis by cryo-EM revealed that CA521FALA recognizes an epitope overlapping with angiotensin converting enzyme 2 (ACE2)-binding sites in SARS-CoV-2 RBD in the Spike protein. CA521FALA blocks the interaction by binding all three RBDs of one SARS-CoV-2 spike trimer simultaneously. These results demonstrate the importance for antibody-based therapeutic interventions against COVID-19 and identifies CA521FALA a promising antibody that reacts with SARS-CoV-2 Spike protein to strongly neutralize its activity.


Assuntos
Anticorpos Monoclonais/farmacologia , Anticorpos Neutralizantes/farmacologia , COVID-19/prevenção & controle , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Enzima de Conversão de Angiotensina 2/imunologia , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , COVID-19/epidemiologia , COVID-19/virologia , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Pandemias , Ligação Proteica/efeitos dos fármacos , Receptores Virais/imunologia , Receptores Virais/metabolismo , SARS-CoV-2/metabolismo , SARS-CoV-2/fisiologia , Glicoproteína da Espícula de Coronavírus/metabolismo
17.
Front Immunol ; 12: 603563, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33603759

RESUMO

The high infection rate and rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) make it a world-wide pandemic. Individuals infected by the virus exhibited different degrees of symptoms, and most convalescent individuals have been shown to develop both cellular and humoral immune responses. However, virus-specific adaptive immune responses in severe patients during acute phase have not been thoroughly studied. Here, we found that in a group of COVID-19 patients with acute respiratory distress syndrome (ARDS) during hospitalization, most of them mounted SARS-CoV-2-specific antibody responses, including neutralizing antibodies. However, compared to healthy controls, the percentages and absolute numbers of both NK cells and CD8+ T cells were significantly reduced, with decreased IFNγ expression in CD4+ T cells in peripheral blood from severe patients. Most notably, their peripheral blood lymphocytes failed in producing IFNγ against viral proteins. Thus, severe COVID-19 patients at acute infection stage developed SARS-CoV-2-specific antibody responses but were impaired in cellular immunity, which emphasizes on the role of cellular immunity in COVID-19.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , COVID-19/imunologia , Células Matadoras Naturais/imunologia , Síndrome do Desconforto Respiratório/imunologia , SARS-CoV-2/imunologia , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Contagem de Células , Células Cultivadas , Progressão da Doença , Feminino , Humanos , Imunidade Celular , Interferon gama/metabolismo , Masculino , Pessoa de Meia-Idade
18.
Cell Res ; 31(4): 404-414, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33510385

RESUMO

The newly identified Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has resulted in a global health emergency because of its rapid spread and high mortality. The molecular mechanism of interaction between host and viral genomic RNA is yet unclear. We demonstrate herein that SARS-CoV-2 genomic RNA, as well as the negative-sense RNA, is dynamically N6-methyladenosine (m6A)-modified in human and monkey cells. Combined RIP-seq and miCLIP analyses identified a total of 8 m6A sites at single-base resolution in the genome. Especially, epidemic strains with mutations at these identified m6A sites have emerged worldwide, and formed a unique cluster in the US as indicated by phylogenetic analysis. Further functional experiments showed that m6A methylation negatively regulates SARS-CoV-2 infection. SARS-CoV-2 infection also triggered a global increase in host m6A methylome, exhibiting altered localization and motifs of m6A methylation in mRNAs. Altogether, our results identify m6A as a dynamic epitranscriptomic mark mediating the virus-host interaction.


Assuntos
Adenosina/análogos & derivados , Genoma Viral , SARS-CoV-2/genética , Adenosina/metabolismo , Animais , COVID-19/patologia , COVID-19/virologia , Linhagem Celular , Chlorocebus aethiops , Metilação de DNA , Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno , Humanos , Mutagênese Sítio-Dirigida , Filogenia , RNA Mensageiro/genética , SARS-CoV-2/classificação , SARS-CoV-2/isolamento & purificação , SARS-CoV-2/fisiologia , Células Vero , Replicação Viral
19.
Cell Res ; 31(1): 25-36, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33262452

RESUMO

Structural principles underlying the composition and synergistic mechanisms of protective monoclonal antibody cocktails are poorly defined. Here, we exploited antibody cooperativity to develop a therapeutic antibody cocktail against SARS-CoV-2. On the basis of our previously identified humanized cross-neutralizing antibody H014, we systematically analyzed a fully human naive antibody library and rationally identified a potent neutralizing antibody partner, P17, which confers effective protection in animal model. Cryo-EM studies dissected the nature of the P17 epitope, which is SARS-CoV-2 specific and distinctly different from that of H014. High-resolution structure of the SARS-CoV-2 spike in complex with H014 and P17, together with functional investigations revealed that in a two-antibody cocktail, synergistic neutralization was achieved by S1 shielding and conformational locking, thereby blocking receptor attachment and viral membrane fusion, conferring high potency as well as robustness against viral mutation escape. Furthermore, cluster analysis identified a hypothetical 3rd antibody partner for further reinforcing the cocktail as pan-SARS-CoVs therapeutics.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , COVID-19 , Epitopos/imunologia , SARS-CoV-2/imunologia , Anticorpos de Cadeia Única/imunologia , Animais , Anticorpos Neutralizantes/farmacologia , Anticorpos Antivirais/farmacologia , COVID-19/imunologia , COVID-19/prevenção & controle , Chlorocebus aethiops , Modelos Animais de Doenças , Humanos , Anticorpos de Cadeia Única/farmacologia , Células Vero
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