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1.
J Virol ; : e0087922, 2022 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-36377874

RESUMO

The glycan loop of Zika virus (ZIKV) envelope protein (E) contains the glycosylation site and has been well documented to be important for viral pathogenesis and transmission. In the present study, we report that deletions in the E glycan loop, which were recorded in African ZIKV strains previously, have re-emerged in their contemporary Asian lineages. Here, we generated recombinant ZIKV containing specific deletions in the E glycan loop by reverse genetics. Extensive in vitro and in vivo characterization of these deletion mutants demonstrated an attenuated phenotype in an adult A129 mouse model and reduced oral infections in mosquitoes. Surprisingly, these glycan loop deletion mutants exhibited an enhanced neurovirulence phenotype, and resulted in a more severe microcephalic brain in neonatal mouse models. Crystal structures of the ZIKV E protein and a deletion mutant at 2.5 and 2.6 Å, respectively, revealed that deletion of the glycan loop induces encephalitic flavivirus-like conformational alterations, including the appearance of perforations on the surface and a clear change in the topology of the loops. Overall, our results demonstrate that the E glycan loop deletions represent neonatal mouse neurovirulence markers of ZIKV. IMPORTANCE Zika virus (ZIKV) has been identified as a cause of microcephaly and acquired evolutionary mutations since its discovery. Previously deletions in the E glycan loop were recorded in African ZIKV strains, which have re-emerged in the contemporary Asian lineages recently. The glycan loop deletion mutants are not glycosylated, which are attenuated in adult A129 mouse model and reduced oral infections in mosquitoes. More importantly, the glycan loop deletion mutants induce an encephalitic flavivirus-like conformational alteration in the E homodimer, resulting in a significant enhancement of neonatal mouse neurovirulence. This study underscores the critical role of glycan loop deletion mutants in ZIKV pathogenesis, highlighting a need for global virological surveillance for such ZIKV variants.

2.
J Med Virol ; : e28290, 2022 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-36367083

RESUMO

The geographic range of Zika virus (ZIKV) has expanded from Asia to the Americas, leading to the 2015-2016 pandemic with enhanced neurovirulence. At present, ZIKV is continuously circulating in many Southeast Asian countries. Unfortunately, the persistent evolution of ZIKV in Southeast Asia and its influence on the biological characteristics of the virus remain incompletely understood. In this study, the in vitro and in vivo properties of a new ZIKV isolate obtained from Cambodia in 2019 (CAM/2019) were characterized and compared with those of the Cambodian strain (CAM/2010). Compared with CAM/2010, the CAM/2019 virus showed similar plaque morphology and growth curves in cell cultures and induced comparable viremia and organ viral loads profiles in both BALB/c and A129 (IFNAR1-/- ) mice upon intraperitoneal (i.p.) inoculation. Remarkably, the CAM/2019 virus exhibited enhanced neurovirulence in neonatal mice compared with CAM/2010, with a 74-fold reduction in the 50% lethal dose (LD50 ). Consistently, CAM/2019 produced higher viral loads in the brains of BALB/c neonatal mice than CAM/2010 did. Sequence alignment showed that the CAM/2019 virus has acquired 12 amino acid substitutions, several of which were found to be associated with neurovirulence. In particular, the CAM/2019 virus shared an A1204T substitution in NS2A with the Thai isolate SI-BKK02 that was isolated from a microcephaly case. Taken together, our results indicate that a ZIKV strain isolated with specific mutations has emerged in Cambodia, highlighting the need for extensive molecular and disease surveillance in Cambodia and other Asian countries.

3.
Emerg Microbes Infect ; 11(1): 2350-2358, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36069671

RESUMO

Zika virus (ZIKV) is primarily transmitted through mosquito bites and sexual contact, and vertical transmission of ZIKV has also been observed in humans. In addition, ZIKV infection via unknown transmission routes has been frequently reported in clinical settings. However, whether ZIKV can be transmitted via aerosol routes remains unknown. In this study, we demonstrated that aerosolized ZIKV is fully infectious in vitro and in vivo. Remarkably, intratracheal (i.t.) inoculation with aerosolized ZIKV led to rapid viremia and viral secretion in saliva, as well as robust humoral and innate immune responses in guinea pigs. Transcriptome analysis further revealed that the expression of genes related to viral processes, biological regulation and the immune response was significantly changed. Together, our results confirm that aerosolized ZIKV can result in systemic infection and induce both innate and adaptive immune responses in guinea pigs, highlighting the possibility of ZIKV transmission via aerosols.


Assuntos
Infecção por Zika virus , Zika virus , Animais , Cobaias , Humanos , Imunidade Humoral , Transmissão Vertical de Doenças Infecciosas , Viremia , Zika virus/fisiologia
4.
Microbiol Spectr ; 10(5): e0224622, 2022 Oct 26.
Artigo em Inglês | MEDLINE | ID: mdl-35980184

RESUMO

The yellow fever (YF) live attenuated vaccine strain 17D (termed 17D) has been widely used for the prevention and control of YF disease. However, 17D retains significant neurovirulence and viscerotropism in mice, which is probably linked to the increased occurrences of serious adverse events following 17D vaccination. Thus, the development of an updated version of the YF vaccine with an improved safety profile is of high priority. Here, we generated a viable bicistronic YF virus (YFV) by incorporating the internal ribosome entry site (IRES) from Encephalomyocarditis virus into an infectious clone of YFV 17D. The resulting recombinant virus, 17D-IRES, exhibited similar replication efficiency to its parental virus (17D) in mammalian cell lines, while it was highly restricted in mosquito cells. Serial passage of 17D-IRES in BHK-21 cells showed good genetic stability. More importantly, in comparison with the parental 17D, 17D-IRES displayed significantly decreased mouse neurovirulence and viscerotropism in type I interferon (IFN)-signaling-deficient and immunocompetent mouse models. Interestingly, 17D-IRES showed enhanced sensitivity to type I IFN compared with 17D. Moreover, immunization with 17D-IRES provided solid protection for mice against a lethal challenge with YFV. These preclinical data support further development of 17D-IRES as an updated version for the approved YF vaccine. This IRES-based attenuation strategy could be also applied to the design of live attenuated vaccines against other mosquito-borne flaviviruses. IMPORTANCE Yellow fever (YF) continually spreads and causes epidemics around the world, posing a great threat to human health. The YF live attenuated vaccine 17D is considered the most efficient vaccine available and helps to successfully control disease epidemics. However, side effects may occur after vaccination, such as viscerotropic disease (YEL-AVD) and neurotropic adverse disease (YEL-AND). Thus, there is an urgent need for a safer YF vaccine. Here, an IRES strategy was employed, and a bicistronic YFV was successfully developed (named 17D-IRES). 17D-IRES showed effective replication and genetic stability in vitro and high attenuation in vivo. Importantly, 17D-IRES induced humoral and cellular immune responses and conferred full protection against lethal YFV challenge. Our study provides data suggesting that 17D-IRES, with its prominent advantages, could be a vaccine candidate against YF. Moreover, this IRES-based bicistronic technology platform represents a promising strategy for developing other live attenuated vaccines against emerging viruses.


Assuntos
Interferon Tipo I , Vacina contra Febre Amarela , Febre Amarela , Camundongos , Humanos , Animais , Febre Amarela/prevenção & controle , Vacinas Atenuadas/efeitos adversos , Vacinas Atenuadas/genética , Sítios Internos de Entrada Ribossomal , Vacina contra Febre Amarela/efeitos adversos , Vacina contra Febre Amarela/genética , Vírus da Febre Amarela/genética , Antígenos Virais , Interferon Tipo I/genética , Mamíferos/genética
5.
Cell Biosci ; 12(1): 139, 2022 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-36042495

RESUMO

BACKGROUND: Recognition of viral invasion by innate antiviral immune system triggers activation of the type I interferon (IFN-I) and proinflammatory signaling pathways. Subsequently, IFN-I induction regulates expression of a group of genes known as IFN-I-stimulated genes (ISGs) to block viral infection. The tripartite motif containing 22 (TRIM22) is an ISG with strong antiviral functions. RESULTS: Here we have shown that the TRIM22 has been strongly upregulated both transcriptionally and translationally upon Zika virus (ZIKV) infection. ZIKV infection is associated with a wide range of clinical manifestations in human from mild to severe symptoms including abnormal fetal brain development. We found that the antiviral function of TRIM22 plays a crucial role in counterattacking ZIKV infection. Overexpression of TRIM22 protein inhibited ZIKV growth whereas deletion of TRIM22 in host cells increased ZIKV infectivity. Mechanistically, TRIM22, as a functional E3 ubiquitin ligase, promoted the ubiquitination and degradation of ZIKV nonstructural protein 1 (NS1) and nonstructural protein 3 (NS3). Further studies showed that the SPRY domain and Ring domain of TRIM22 played important roles in protein interaction and degradation, respectively. In addition, we found that TRIM22 also inhibited other flaviviruses infection including dengue virus (DENV) and yellow fever virus (YFV). CONCLUSION: Thus, TRIM22 is an ISG with important role in host defense against flaviviruses through binding and degradation of the NS1 and NS3 proteins.

6.
J Mater Chem B ; 10(28): 5454-5464, 2022 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-35786741

RESUMO

The SARS-CoV-2 pandemic has become a severe global public health event, and the development of protective and therapeutic strategies is urgently needed. Downregulation of angiotensin converting enzyme 2 (ACE2; one of the important SARS-CoV-2 entry receptors) and aberrant inflammatory responses (cytokine storm) are the main targets to inhibit and control COVID-19 invasion. Silver nanomaterials have well-known pharmaceutical properties, including antiviral, antibacterial, and anticancer properties. Here, based on a self-established metal evaporation-condensation-size graded collection system, smaller silver particles reaching the Ångstrom scale (AgÅPs) were fabricated and coated with fructose to obtain a stabilized AgÅP solution (F-AgÅPs). F-AgÅPs potently inactivated SARS-CoV-2 and prevented viral infection. Considering the application of anti-SARS-CoV-2, a sterilized F-AgÅP solution was produced via spray formulation. In our model, the F-AgÅP spray downregulated ACE2 expression and attenuated proinflammatory factors. Moreover, F-AgÅPs were found to be rapidly eliminated to avoid respiratory and systemic toxicity in this study as well as our previous studies. This work presents a safe and potent anti-SARS-CoV-2 agent using an F-AgÅP spray.


Assuntos
Enzima de Conversão de Angiotensina 2 , Humanos , Peptidil Dipeptidase A/metabolismo , SARS-CoV-2 , Prata/farmacologia
7.
Cell Biosci ; 12(1): 63, 2022 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-35581593

RESUMO

BACKGROUND: Neutralizing antibodies are approved drugs to treat coronavirus disease-2019 (COVID-19) patients, yet mutations in severe acute respiratory syndrome coronavirus (SARS-CoV-2) variants may reduce the antibody neutralizing activity. New monoclonal antibodies (mAbs) and antibody remolding strategies are recalled in the battle with COVID-19 epidemic. RESULTS: We identified multiple mAbs from antibody phage display library made from COVID-19 patients and further characterized the R3P1-E4 clone, which effectively suppressed SARS-CoV-2 infection and rescued the lethal phenotype in mice infected with SARS-CoV-2. Crystal structural analysis not only explained why R3P1-E4 had selectively reduced binding and neutralizing activity to SARS-CoV-2 variants carrying K417 mutations, but also allowed us to engineer mutant antibodies with improved neutralizing activity against these variants. Thus, we screened out R3P1-E4 mAb which inhibits SARS-CoV-2 and related mutations in vitro and in vivo. Antibody engineering improved neutralizing activity of R3P1-E4 against K417 mutations. CONCLUSION: Our studies have outlined a strategy to identify and engineer neutralizing antibodies against SARS-CoV-2 variants.

8.
Innovation (Camb) ; 3(2): 100221, 2022 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-35252935

RESUMO

The highly pathogenic and readily transmissible SARS-CoV-2 has caused a global coronavirus pandemic, urgently requiring effective countermeasures against its rapid expansion. All available vaccine platforms are being used to generate safe and effective COVID-19 vaccines. Here, we generated a live-attenuated candidate vaccine strain by serial passaging of a SARS-CoV-2 clinical isolate in Vero cells. Deep sequencing revealed the dynamic adaptation of SARS-CoV-2 in Vero cells, resulting in a stable clone with a deletion of seven amino acids (N679SPRRAR685) at the S1/S2 junction of the S protein (named VAS5). VAS5 showed significant attenuation of replication in multiple human cell lines, human airway epithelium organoids, and hACE2 mice. Viral fitness competition assays demonstrated that VAS5 showed specific tropism to Vero cells but decreased fitness in human cells compared with the parental virus. More importantly, a single intranasal injection of VAS5 elicited a high level of neutralizing antibodies and prevented SARS-CoV-2 infection in mice as well as close-contact transmission in golden Syrian hamsters. Structural and biochemical analysis revealed a stable and locked prefusion conformation of the S trimer of VAS5, which most resembles SARS-CoV-2-3Q-2P, an advanced vaccine immunogen (NVAX-CoV2373). Further systematic antigenic profiling and immunogenicity validation confirmed that the VAS5 S trimer presents an enhanced antigenic mimic of the wild-type S trimer. Our results not only provide a potent live-attenuated vaccine candidate against COVID-19 but also clarify the molecular and structural basis for the highly attenuated and super immunogenic phenotype of VAS5.

9.
Cell Res ; 32(4): 375-382, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35210606

RESUMO

Monoclonal antibodies represent important weapons in our arsenal to against the COVID-19 pandemic. However, this potential is severely limited by the time-consuming process of developing effective antibodies and the relative high cost of manufacturing. Herein, we present a rapid and cost-effective lipid nanoparticle (LNP) encapsulated-mRNA platform for in vivo delivery of SARS-CoV-2 neutralization antibodies. Two mRNAs encoding the light and heavy chains of a potent SARS-CoV-2 neutralizing antibody HB27, which is currently being evaluated in clinical trials, were encapsulated into clinical grade LNP formulations (named as mRNA-HB27-LNP). In vivo characterization demonstrated that intravenous administration of mRNA-HB27-LNP in mice resulted in a longer circulating half-life compared with the original HB27 antibody in protein format. More importantly, a single prophylactic administration of mRNA-HB27-LNP provided protection against SARS-CoV-2 challenge in mice at 1, 7 and even 63 days post administration. In a close contact transmission model, prophylactic administration of mRNA-HB27-LNP prevented SARS-CoV-2 infection between hamsters in a dose-dependent manner. Overall, our results demonstrate a superior long-term protection against SARS-CoV-2 conferred by a single administration of this unique mRNA antibody, highlighting the potential of this universal platform for antibody-based disease prevention and therapy against COVID-19 as well as a variety of other infectious diseases.


Assuntos
COVID-19 , SARS-CoV-2 , Animais , Anticorpos Neutralizantes/uso terapêutico , Anticorpos Antivirais/uso terapêutico , COVID-19/prevenção & controle , Cricetinae , Humanos , Lipossomos , Camundongos , Nanopartículas , Pandemias/prevenção & controle , RNA Mensageiro/genética , Glicoproteína da Espícula de Coronavírus
10.
Lancet Microbe ; 3(3): e193-e202, 2022 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-35098177

RESUMO

BACKGROUND: Safe and effective vaccines are urgently needed to end the COVID-19 pandemic caused by SARS-CoV-2 infection. We aimed to assess the preliminary safety, tolerability, and immunogenicity of an mRNA vaccine ARCoV, which encodes the SARS-CoV-2 spike protein receptor-binding domain (RBD). METHODS: This single centre, double-blind, randomised, placebo-controlled, dose-escalation, phase 1 trial of ARCoV was conducted at Shulan (Hangzhou) hospital in Hangzhou, Zhejiang province, China. Healthy adults aged 18-59 years negative for SARS-CoV-2 infection were enrolled and randomly assigned using block randomisation to receive an intramuscular injection of vaccine or placebo. Vaccine doses were 5 µg, 10 µg, 15 µg, 20 µg, and 25 µg. The first six participants in each block were sentinels and along with the remaining 18 participants, were randomly assigned to groups (5:1). In block 1 sentinels were given the lowest vaccine dose and after a 4-day observation with confirmed safety analyses, the remaining 18 participants in the same dose group proceeded and sentinels in block 2 were given their first administration on a two-dose schedule, 28 days apart. All participants, investigators, and staff doing laboratory analyses were masked to treatment allocation. Humoral responses were assessed by measuring anti-SARS-CoV-2 RBD IgG using a standardised ELISA and neutralising antibodies using pseudovirus-based and live SARS-CoV-2 neutralisation assays. SARS-CoV-2 RBD-specific T-cell responses, including IFN-γ and IL-2 production, were assessed using an enzyme-linked immunospot (ELISpot) assay. The primary outcome for safety was incidence of adverse events or adverse reactions within 60 min, and at days 7, 14, and 28 after each vaccine dose. The secondary safety outcome was abnormal changes detected by laboratory tests at days 1, 4, 7, and 28 after each vaccine dose. For immunogenicity, the secondary outcome was humoral immune responses: titres of neutralising antibodies to live SARS-CoV-2, neutralising antibodies to pseudovirus, and RBD-specific IgG at baseline and 28 days after first vaccination and at days 7, 15, and 28 after second vaccination. The exploratory outcome was SARS-CoV-2-specific T-cell responses at 7 days after the first vaccination and at days 7 and 15 after the second vaccination. This trial is registered with www.chictr.org.cn (ChiCTR2000039212). FINDINGS: Between Oct 30 and Dec 2, 2020, 230 individuals were screened and 120 eligible participants were randomly assigned to receive five-dose levels of ARCoV or a placebo (20 per group). All participants received the first vaccination and 118 received the second dose. No serious adverse events were reported within 56 days after vaccination and the majority of adverse events were mild or moderate. Fever was the most common systemic adverse reaction (one [5%] of 20 in the 5 µg group, 13 [65%] of 20 in the 10 µg group, 17 [85%] of 20 in the 15 µg group, 19 [95%] of 20 in the 20 µg group, 16 [100%] of 16 in the 25 µg group; p<0·0001). The incidence of grade 3 systemic adverse events were none (0%) of 20 in the 5 µg group, three (15%) of 20 in the 10 µg group, six (30%) of 20 in the 15 µg group, seven (35%) of 20 in the 20 µg group, five (31%) of 16 in the 25 µg group, and none (0%) of 20 in the placebo group (p=0·0013). As expected, the majority of fever resolved in the first 2 days after vaccination for all groups. The incidence of solicited systemic adverse events was similar after administration of ARCoV as a first or second vaccination. Humoral immune responses including anti-RBD IgG and neutralising antibodies increased significantly 7 days after the second dose and peaked between 14 and 28 days thereafter. Specific T-cell response peaked between 7 and 14 days after full vaccination. 15 µg induced the highest titre of neutralising antibodies, which was about twofold more than the antibody titre of convalescent patients with COVID-19. INTERPRETATION: ARCoV was safe and well tolerated at all five doses. The acceptable safety profile, together with the induction of strong humoral and cellular immune responses, support further clinical testing of ARCoV at a large scale. FUNDING: National Key Research and Development Project of China, Academy of Medical Sciences China, National Natural Science Foundation China, and Chinese Academy of Medical Sciences.


Assuntos
COVID-19 , SARS-CoV-2 , Adulto , Anticorpos Neutralizantes , Anticorpos Antivirais , COVID-19/prevenção & controle , Vacinas contra COVID-19/efeitos adversos , China , Humanos , Imunogenicidade da Vacina , Imunoglobulina G , Pandemias/prevenção & controle , Glicoproteína da Espícula de Coronavírus , Vacinas Sintéticas , Vacinas de mRNA
11.
J Craniofac Surg ; 33(5): e467-e470, 2022.
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.


Assuntos
Luxações Articulares , Transtornos da Articulação Temporomandibular , Osso e Ossos , Humanos , Luxações Articulares/diagnóstico por imagem , Luxações Articulares/terapia , Imageamento por Ressonância Magnética , Placas Oclusais , Contenções , Disco da Articulação Temporomandibular , Transtornos da Articulação Temporomandibular/terapia , Resultado do Tratamento
12.
J Craniofac Surg ; 33(4): 1104-1107, 2022 Jun 01.
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.


Assuntos
Luxações Articulares , Ritidoplastia , Disco da Articulação Temporomandibular , Transtornos da Articulação Temporomandibular , Feminino , Humanos , Luxações Articulares/diagnóstico por imagem , Luxações Articulares/cirurgia , Imageamento por Ressonância Magnética , Pessoa de Meia-Idade , Ritidoplastia/métodos , Disco da Articulação Temporomandibular/diagnóstico por imagem , Disco da Articulação Temporomandibular/cirurgia , Transtornos da Articulação Temporomandibular/diagnóstico por imagem , Transtornos da Articulação Temporomandibular/cirurgia , Resultado do Tratamento
13.
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 , Vacinas de mRNA/farmacologia , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , COVID-19/prevenção & controle , Vacinas contra COVID-19/imunologia , Chlorocebus aethiops , Humanos , Macaca fascicularis , Células Vero , Vacinas de mRNA/imunologia
17.
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
18.
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.

19.
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.

20.
Virol Sin ; 36(6): 1484-1491, 2021 Dec.
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.


Assuntos
COVID-19 , Anticorpos Neutralizantes , Anticorpos Antivirais/metabolismo , Escherichia coli/metabolismo , Humanos , Leucócitos Mononucleares/metabolismo , Ligação Proteica , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus/metabolismo
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