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1.
BMC Immunol ; 22(1): 22, 2021 03 25.
Artículo en Inglés | MEDLINE | ID: mdl-33765919

RESUMEN

BACKGROUND: The spread of a novel coronavirus termed severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in China and other countries is of great concern worldwide with no effective vaccine. This study aimed to design a novel vaccine construct against SARS-CoV-2 from the spike S protein and orf1ab polyprotein using immunoinformatics tools. The vaccine was designed from conserved epitopes interacted against B and T lymphocytes by the combination of highly immunogenic epitopes with suitable adjuvant and linkers. RESULTS: The proposed vaccine composed of 526 amino acids and was shown to be antigenic in Vaxigen server (0.6194) and nonallergenic in Allertop server. The physiochemical properties of the vaccine showed isoelectric point of 10.19. The instability index (II) was 31.25 classifying the vaccine as stable. Aliphatic index was 84.39 and the grand average of hydropathicity (GRAVY) was - 0.049 classifying the vaccine as hydrophilic. Vaccine tertiary structure was predicted, refined and validated to assess the stability of the vaccine via Ramachandran plot and ProSA-web servers. Moreover, solubility of the vaccine construct was greater than the average solubility provided by protein sol and SOLpro servers indicating the solubility of the vaccine construct. Disulfide engineering was performed to reduce the high mobile regions in the vaccine to enhance stability. Docking of the vaccine construct with TLR4 demonstrated efficient binding energy with attractive binding energy of - 338.68 kcal/mol and - 346.89 kcal/mol for TLR4 chain A and chain B respectively. Immune simulation significantly provided high levels of immunoglobulins, T-helper cells, T-cytotoxic cells and INF-γ. Upon cloning, the vaccine protein was reverse transcribed into DNA sequence and cloned into pET28a(+) vector to ensure translational potency and microbial expression. CONCLUSION: A unique vaccine construct from spike S protein and orf1ab polyprotein against B and T lymphocytes was generated with potential protection against the pandemic. The present study might assist in developing a suitable therapeutics protocol to combat SARSCoV-2 infection.


Asunto(s)
/inmunología , Epítopos de Linfocito B , Epítopos de Linfocito T , Glicoproteína de la Espiga del Coronavirus , Proteínas Virales , Linfocitos B/inmunología , /química , /inmunología , Epítopos de Linfocito B/química , Epítopos de Linfocito B/genética , Epítopos de Linfocito B/inmunología , Epítopos de Linfocito T/química , Epítopos de Linfocito T/genética , Epítopos de Linfocito T/inmunología , Humanos , Poliproteínas/química , Poliproteínas/genética , Poliproteínas/inmunología , /genética , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/inmunología , Linfocitos T/inmunología , Proteínas Virales/química , Proteínas Virales/genética , Proteínas Virales/inmunología
2.
Biosens Bioelectron ; 180: 113122, 2021 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-33706157

RESUMEN

As the COVID-19 pandemic continues, there is an imminent need for rapid diagnostic tools and effective antivirals targeting SARS-CoV-2. We have developed a novel bioluminescence-based biosensor to probe a key host-virus interaction during viral entry: the binding of SARS-CoV-2 viral spike (S) protein to its receptor, angiotensin-converting enzyme 2 (ACE2). Derived from Nanoluciferase binary technology (NanoBiT), the biosensor is composed of Nanoluciferase split into two complementary subunits, Large BiT and Small BiT, fused to the Spike S1 domain of the SARS-CoV-2 S protein and ACE2 ectodomain, respectively. The ACE2-S1 interaction results in reassembly of functional Nanoluciferase, which catalyzes a bioluminescent reaction that can be assayed in a highly sensitive and specific manner. We demonstrate the biosensor's large dynamic range, enhanced thermostability and pH tolerance. In addition, we show the biosensor's versatility towards the high-throughput screening of drugs which disrupt the ACE2-S1 interaction, as well as its ability to act as a surrogate virus neutralization assay. Results obtained with our biosensor correlate well with those obtained with a Spike-pseudotyped lentivirus assay. This rapid in vitro tool does not require infectious virus and should enable the timely development of antiviral modalities targeting SARS-CoV-2 entry.


Asunto(s)
/química , Técnicas Biosensibles/métodos , Mediciones Luminiscentes/métodos , Glicoproteína de la Espiga del Coronavirus/química , Células HEK293 , Humanos , Luciferasas , Pruebas de Neutralización , Internalización del Virus
3.
J Phys Chem B ; 125(10): 2533-2550, 2021 03 18.
Artículo en Inglés | MEDLINE | ID: mdl-33657325

RESUMEN

The novel RNA virus, severe acute respiratory syndrome coronavirus II (SARS-CoV-2), is currently the leading cause of mortality in 2020, having led to over 1.6 million deaths and infecting over 75 million people worldwide by December 2020. While vaccination has started and several clinical trials for a number of vaccines are currently underway, there is a pressing need for a cure for those already infected with the virus. Of particular interest in the design of anti-SARS-CoV-2 therapeutics is the human protein angiotensin converting enzyme II (ACE2) to which this virus adheres before entry into the host cell. The SARS-CoV-2 virion binds to cell-surface bound ACE2 via interactions of the spike protein (s-protein) on the viral surface with ACE2. In this paper, we use all-atom molecular dynamics simulations and binding enthalpy calculations to determine the effect that a bound ACE2 active site inhibitor (MLN-4760) would have on the binding affinity of SARS-CoV-2 s-protein with ACE2. Our analysis indicates that the binding enthalpy could be reduced for s-protein adherence to the active site inhibitor-bound ACE2 protein by as much as 1.48-fold as an upper limit. This weakening of binding strength was observed to be due to the destabilization of the interactions between ACE2 residues Glu-35, Glu-37, Tyr-83, Lys-353, and Arg-393 and the SARS-CoV-2 s-protein receptor binding domain (RBD). The conformational changes were shown to lead to weakening of ACE2 interactions with SARS-CoV-2 s-protein, therefore reducing s-protein binding strength. Further, we observed increased conformational lability of the N-terminal helix and a conformational shift of a significant portion of the ACE2 motifs involved in s-protein binding, which may affect the kinetics of the s-protein binding when the small molecule inhibitor is bound to the ACE2 active site. These observations suggest potential new ways for interfering with the SARS-CoV-2 adhesion by modulating ACE2 conformation through distal active site inhibitor binding.


Asunto(s)
/metabolismo , Inhibidores de Proteasas/metabolismo , Glicoproteína de la Espiga del Coronavirus/metabolismo , /antagonistas & inhibidores , Sitios de Unión , /virología , Dominio Catalítico , Diseño de Fármacos , Humanos , Enlace de Hidrógeno , Simulación de Dinámica Molecular , Inhibidores de Proteasas/química , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Estructura Terciaria de Proteína , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/metabolismo , Glicoproteína de la Espiga del Coronavirus/química , Termodinámica
4.
Drug Des Devel Ther ; 15: 1111-1133, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33737804

RESUMEN

Purpose: SARS-CoV-2 engages human ACE2 through its spike (S) protein receptor binding domain (RBD) to enter the host cell. Recent computational studies have reported that withanone and withaferin A, phytochemicals found in Withania somnifera, target viral main protease (MPro) and host transmembrane TMPRSS2, and glucose related protein 78 (GRP78), respectively, implicating their potential as viral entry inhibitors. Absence of specific treatment against SARS-CoV-2 infection has encouraged exploration of phytochemicals as potential antivirals. Aim: This study aimed at in silico exploration, along with in vitro and in vivo validation of antiviral efficacy of the phytochemical withanone. Methods: Through molecular docking, molecular dynamic (MD) simulation and electrostatic energy calculation the plausible biochemical interactions between withanone and the ACE2-RBD complex were investigated. These in silico observations were biochemically validated by ELISA-based assays. Withanone-enriched extract from W. somnifera was tested for its ability to ameliorate clinically relevant pathological features, modelled in humanized zebrafish through SARS-CoV-2 recombinant spike (S) protein induction. Results: Withanone bound efficiently at the interacting interface of the ACE2-RBD complex and destabilized it energetically. The electrostatic component of binding free energies of the complex was significantly decreased. The two intrachain salt bridge interactions (K31-E35) and the interchain long-range ion-pair (K31-E484), at the ACE2-RBD interface were completely abolished by withanone, in the 50 ns simulation. In vitro binding assay experimentally validated that withanone efficiently inhibited (IC50=0.33 ng/mL) the interaction between ACE2 and RBD, in a dose-dependent manner. A withanone-enriched extract, without any co-extracted withaferin A, was prepared from W. somnifera leaves. This enriched extract was found to be efficient in ameliorating human-like pathological responses induced in humanized zebrafish by SARS-CoV-2 recombinant spike (S) protein. Conclusion: In conclusion, this study provided experimental validation for computational insight into the potential of withanone as a potent inhibitor of SARS-CoV-2 coronavirus entry into the host cells.


Asunto(s)
/metabolismo , Antivirales/farmacología , /efectos de los fármacos , Glicoproteína de la Espiga del Coronavirus/metabolismo , Withania , Witanólidos/farmacología , Células A549 , Animales , Antivirales/química , Antivirales/aislamiento & purificación , /virología , Modelos Animales de Enfermedad , Femenino , Interacciones Huésped-Patógeno , Humanos , Masculino , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Dominios y Motivos de Interacción de Proteínas , /patogenicidad , Glicoproteína de la Espiga del Coronavirus/química , Electricidad Estática , Relación Estructura-Actividad , Internalización del Virus/efectos de los fármacos , Withania/química , Witanólidos/química , Witanólidos/aislamiento & purificación , Pez Cebra
5.
Viruses ; 13(3)2021 02 25.
Artículo en Inglés | MEDLINE | ID: mdl-33669132

RESUMEN

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiologic agent responsible for the recent coronavirus disease 2019 (COVID-19) pandemic. Productive SARS-CoV-2 infection relies on viral entry into cells expressing angiotensin-converting enzyme 2 (ACE2). Indeed, viral entry into cells is mostly mediated by the early interaction between the viral spike protein S and its ACE2 receptor. The S/ACE2 complex is, thus, the first contact point between the incoming virus and its cellular target; consequently, it has been considered an attractive therapeutic target. To further characterize this interaction and the cellular processes engaged in the entry step of the virus, we set up various in silico, in vitro and in cellulo approaches that allowed us to specifically monitor the S/ACE2 association. We report here a computational model of the SARS-CoV-2 S/ACE2 complex, as well as its biochemical and biophysical monitoring using pulldown, AlphaLISA and biolayer interferometry (BLI) binding assays. This led us to determine the kinetic parameters of the S/ACE2 association and dissociation steps. In parallel to these in vitro approaches, we developed in cellulo transduction assays using SARS-CoV-2 pseudotyped lentiviral vectors and HEK293T-ACE2 cell lines generated in-house. This allowed us to recapitulate the early replication stage of the infection mediated by the S/ACE2 interaction and to detect cell fusion induced by the interaction. Finally, a cell imaging system was set up to directly monitor the S/ACE2 interaction in a cellular context and a flow cytometry assay was developed to quantify this association at the cell surface. Together, these different approaches are available for both basic and clinical research, aiming to characterize the entry step of the original SARS-CoV-2 strain and its variants as well as to investigate the possible chemical modulation of this interaction. All these models will help in identifying new antiviral agents and new chemical tools for dissecting the virus entry step.


Asunto(s)
/metabolismo , /fisiología , Glicoproteína de la Espiga del Coronavirus/metabolismo , Internalización del Virus , /química , Simulación por Computador , Células HEK293 , Humanos , Técnicas In Vitro , Cinética , Modelos Moleculares , Simulación de Dinámica Molecular , Unión Proteica , Dominios Proteicos , Glicoproteína de la Espiga del Coronavirus/química
8.
J Am Chem Soc ; 143(10): 3959-3966, 2021 03 17.
Artículo en Inglés | MEDLINE | ID: mdl-33657316

RESUMEN

The heterogeneity associated with glycosylation of the 66 N-glycan sites on the protein trimer making up the spike (S) region of the SARS-CoV-2 virus has been assessed by charge detection mass spectrometry (CDMS). CDMS allows simultaneous measurement of the mass-to-charge ratio and charge of individual ions, so that mass distributions can be determined for highly heterogeneous proteins such as the heavily glycosylated S protein trimer. The CDMS results are compared to recent glycoproteomics studies of the structure and abundance of glycans at specific sites. Interestingly, average glycan masses determined by "top-down" CDMS measurements are 35-47% larger than those obtained from the "bottom-up" glycoproteomics studies, suggesting that the glycoproteomic measurements underestimated the abundances of larger, more-complex glycans. Moreover, the distribution of glycan masses determined by CDMS is much broader than the distribution expected from the glycoproteomics studies, assuming that glycan processing on each trimer is not correlated. The breadth of the glycan mass distribution therefore indicates heterogeneity in the extent of glycan processing of the S protein trimers, with some trimers being much more heavily processed than others. This heterogeneity may have evolved as a way of further confounding the host's immune system.


Asunto(s)
Espectrometría de Masas , Polisacáridos/metabolismo , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/metabolismo , Células HEK293 , Humanos , Dominios Proteicos
9.
mBio ; 12(2)2021 03 02.
Artículo en Inglés | MEDLINE | ID: mdl-33653892

RESUMEN

The spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been identified as the prime target for vaccine development. The spike protein mediates both binding to host cells and membrane fusion and is also so far the only known viral target of neutralizing antibodies. Coronavirus spike proteins are large trimers that are relatively unstable, a feature that might be enhanced by the presence of a polybasic cleavage site in SARS-CoV-2 spike. Exchange of K986 and V987 for prolines has been shown to stabilize the trimers of SARS-CoV-1 and the Middle East respiratory syndrome coronavirus spike proteins. Here, we test multiple versions of a soluble spike protein for their immunogenicity and protective effect against SARS-CoV-2 challenge in a mouse model that transiently expresses human angiotensin-converting enzyme 2 via adenovirus transduction. Variants tested include spike proteins with a deleted polybasic cleavage site, proline mutations, or a combination thereof, besides the wild-type protein. While all versions of the protein were able to induce neutralizing antibodies, only the antigen with both a deleted cleavage site and the K986P and V987P (PP) mutations completely protected from challenge in this mouse model.IMPORTANCE A vaccine for SARS-CoV-2 is urgently needed. A better understanding of antigen design and attributes that vaccine candidates need to have to induce protective immunity is of high importance. The data presented here validate the choice of antigens that contain the PP mutations and suggest that deletion of the polybasic cleavage site may lead to a further-optimized design.


Asunto(s)
Prolina/química , /inmunología , Animales , /inmunología , Ratones , Mutación , Glicoproteína de la Espiga del Coronavirus/química
10.
Biomolecules ; 11(2)2021 02 17.
Artículo en Inglés | MEDLINE | ID: mdl-33671255

RESUMEN

SARS-CoV-2, or COVID-19, has a devastating effect on our society, both in terms of quality of life and death rates; hence, there is an urgent need for developing safe and effective therapeutics against SARS-CoV-2. The most promising strategy to fight against this deadly virus is to develop an effective vaccine. Internalization of SARS-CoV-2 into the human host cell mainly occurs through the binding of the coronavirus spike protein (a trimeric surface glycoprotein) to the human angiotensin-converting enzyme 2 (ACE2) receptor. The spike-ACE2 protein-protein interaction is mediated through the receptor-binding domain (RBD) of the spike protein. Mutations in the spike RBD can significantly alter interactions with the ACE2 host receptor. Due to its important role in virus transmission, the spike RBD is considered to be one of the key molecular targets for vaccine development. In this study, a spike RBD-based subunit vaccine was designed by utilizing a ferritin protein nanocage as a scaffold. Several fusion protein constructs were designed in silico by connecting the spike RBD via a synthetic linker (different sizes) to different ferritin subunits (H-ferritin and L-ferritin). The stability and the dynamics of the engineered nanocage constructs were tested by extensive molecular dynamics simulation (MDS). Based on our MDS analysis, a five amino acid-based short linker (S-Linker) was the most effective for displaying the spike RBD over the surface of ferritin. The behavior of the spike RBD binding regions from the designed chimeric nanocages with the ACE2 receptor was highlighted. These data propose an effective multivalent synthetic nanocage, which might form the basis for new vaccine therapeutics designed against viruses such as SARS-CoV-2.


Asunto(s)
/química , Ferritinas/química , Nanoestructuras/química , Glicoproteína de la Espiga del Coronavirus/química , /metabolismo , /metabolismo , Ferritinas/metabolismo , Humanos , Simulación de Dinámica Molecular , Conformación Proteica , Dominios Proteicos , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Glicoproteína de la Espiga del Coronavirus/metabolismo , Vacunas de Subunidad/química , Vacunas de Subunidad/metabolismo
12.
Sci Rep ; 11(1): 6248, 2021 03 18.
Artículo en Inglés | MEDLINE | ID: mdl-33737523

RESUMEN

The outbreak of a novel febrile respiratory disease called COVID-19, caused by a newfound coronavirus SARS-CoV-2, has brought a worldwide attention. Prioritizing approved drugs is critical for quick clinical trials against COVID-19. In this study, we first manually curated three Virus-Drug Association (VDA) datasets. By incorporating VDAs with the similarity between drugs and that between viruses, we constructed a heterogeneous Virus-Drug network. A novel Random Walk with Restart method (VDA-RWR) was then developed to identify possible VDAs related to SARS-CoV-2. We compared VDA-RWR with three state-of-the-art association prediction models based on fivefold cross-validations (CVs) on viruses, drugs and virus-drug associations on three datasets. VDA-RWR obtained the best AUCs for the three fivefold CVs, significantly outperforming other methods. We found two small molecules coming together on the three datasets, that is, remdesivir and ribavirin. These two chemical agents have higher molecular binding energies of - 7.0 kcal/mol and - 6.59 kcal/mol with the domain bound structure of the human receptor angiotensin converting enzyme 2 (ACE2) and the SARS-CoV-2 spike protein, respectively. Interestingly, for the first time, experimental results suggested that navitoclax could be potentially applied to stop SARS-CoV-2 and remains to further validation.


Asunto(s)
Adenosina Monofosfato/análogos & derivados , Alanina/análogos & derivados , Antivirales/química , Ribavirina/química , Glicoproteína de la Espiga del Coronavirus/química , Adenosina Monofosfato/química , Alanina/química , Compuestos de Anilina/química , Evaluación Preclínica de Medicamentos , Genoma Viral , Simulación del Acoplamiento Molecular , Sulfonamidas/química
13.
Nat Commun ; 12(1): 1577, 2021 03 11.
Artículo en Inglés | MEDLINE | ID: mdl-33707427

RESUMEN

COVID-19 is a severe acute respiratory disease caused by SARS-CoV-2, a new recently emerged sarbecovirus. This virus uses the human ACE2 enzyme as receptor for cell entry, recognizing it with the receptor binding domain (RBD) of the S1 subunit of the viral spike protein. We present the use of phage display to select anti-SARS-CoV-2 spike antibodies from the human naïve antibody gene libraries HAL9/10 and subsequent identification of 309 unique fully human antibodies against S1. 17 antibodies are binding to the RBD, showing inhibition of spike binding to cells expressing ACE2 as scFv-Fc and neutralize active SARS-CoV-2 virus infection of VeroE6 cells. The antibody STE73-2E9 is showing neutralization of active SARS-CoV-2 as IgG and is binding to the ACE2-RBD interface. Thus, universal libraries from healthy human donors offer the advantage that antibodies can be generated quickly and independent from the availability of material from recovering patients in a pandemic situation.


Asunto(s)
/inmunología , Anticuerpos Neutralizantes/genética , Anticuerpos Antivirales/genética , /inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , /química , Animales , Anticuerpos Neutralizantes/aislamiento & purificación , Anticuerpos Antivirales/aislamiento & purificación , Afinidad de Anticuerpos , Línea Celular , Chlorocebus aethiops , Biblioteca de Genes , Voluntarios Sanos , Interacciones Microbiota-Huesped/inmunología , Humanos , Inmunoglobulina G/genética , Inmunoglobulina G/aislamiento & purificación , Modelos Moleculares , Mutación , Pruebas de Neutralización , Pandemias , Biblioteca de Péptidos , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Glicoproteína de la Espiga del Coronavirus/química , Células Vero
14.
Nat Commun ; 12(1): 1607, 2021 03 11.
Artículo en Inglés | MEDLINE | ID: mdl-33707453

RESUMEN

In recognizing the host cellular receptor and mediating fusion of virus and cell membranes, the spike (S) glycoprotein of coronaviruses is the most critical viral protein for cross-species transmission and infection. Here we determined the cryo-EM structures of the spikes from bat (RaTG13) and pangolin (PCoV_GX) coronaviruses, which are closely related to SARS-CoV-2. All three receptor-binding domains (RBDs) of these two spike trimers are in the "down" conformation, indicating they are more prone to adopt the receptor-binding inactive state. However, we found that the PCoV_GX, but not the RaTG13, spike is comparable to the SARS-CoV-2 spike in binding the human ACE2 receptor and supporting pseudovirus cell entry. We further identified critical residues in the RBD underlying different activities of the RaTG13 and PCoV_GX/SARS-CoV-2 spikes. These results collectively indicate that tight RBD-ACE2 binding and efficient RBD conformational sampling are required for the evolution of SARS-CoV-2 to gain highly efficient infection.


Asunto(s)
/virología , Quirópteros/virología , Coronavirus/química , Coronavirus/genética , /química , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/genética , Secuencia de Aminoácidos , Animales , /transmisión , Microscopía por Crioelectrón , Evolución Molecular , Interacciones Microbiota-Huesped , Humanos , Modelos Moleculares , Pandemias , Dominios Proteicos , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Glicoproteína de la Espiga del Coronavirus/ultraestructura
15.
Sci Rep ; 11(1): 5934, 2021 03 15.
Artículo en Inglés | MEDLINE | ID: mdl-33723294

RESUMEN

The aim of this study is to understand adaptive immunity to SARS-CoV-2 through the analysis of B cell epitope and neutralizing activity in coronavirus disease 2019 (COVID-19) patients. We obtained serum from forty-three COVID-19 patients from patients in the intensive care unit of Osaka University Hospital (n = 12) and in Osaka City Juso Hospital (n = 31). Most individuals revealed neutralizing activity against SARS-CoV-2 assessed by a pseudotype virus-neutralizing assay. The antibody production against the spike glycoprotein (S protein) or receptor-binding domain (RBD) of SARS-CoV-2 was elevated, with large individual differences, as assessed by ELISA. We observed the correlation between neutralizing antibody titer and IgG, but not IgM, antibody titer of COVID-19 patients. In the analysis of the predicted the linear B cell epitopes, hot spots in the N-terminal domain of the S protein were observed in the serum from patients in the intensive care unit of Osaka University Hospital. Overall, the analysis of antibody production and B cell epitopes of the S protein from patient serum may provide a novel target for the vaccine development against SARS-CoV-2.


Asunto(s)
/epidemiología , Epítopos de Linfocito B/inmunología , Inmunidad Humoral , /inmunología , Secuencia de Aminoácidos , Anticuerpos Neutralizantes/sangre , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Ensayo de Inmunoadsorción Enzimática , Femenino , Interacciones Huésped-Patógeno , Humanos , Masculino , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/inmunología
16.
mBio ; 12(2)2021 03 16.
Artículo en Inglés | MEDLINE | ID: mdl-33727353

RESUMEN

The angiotensin-converting enzyme 2 (ACE2) receptor is a major severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) host range determinant, and understanding SARS-CoV-2-ACE2 interactions will provide important insights into COVID-19 pathogenesis and animal model development. SARS-CoV-2 cannot infect mice due to incompatibility between its receptor binding domain and the murine ACE2 receptor. Through molecular modeling and empirical in vitro validation, we identified 5 key amino acid differences between murine and human ACE2 that mediate SARS-CoV-2 infection, generating a chimeric humanized murine ACE2. Additionally, we examined the ability of the humanized murine ACE2 receptor to permit infection by an additional preemergent group 2B coronavirus, WIV-1, providing evidence for the potential pan-virus capabilities of this chimeric receptor. Finally, we predicted the ability of these determinants to inform host range identification of preemergent coronaviruses by evaluating hot spot contacts between SARS-CoV-2 and additional potential host receptors. Our results identify residue determinants that mediate coronavirus receptor usage and host range for application in SARS-CoV-2 and emerging coronavirus animal model development.IMPORTANCE SARS-CoV-2 (the causative agent of COVID-19) is a major public health threat and one of two related coronaviruses that have caused epidemics in modern history. A method of screening potential infectible hosts for preemergent and future emergent coronaviruses would aid in mounting rapid response and intervention strategies during future emergence events. Here, we evaluated determinants of SARS-CoV-2 receptor interactions, identifying key changes that enable or prevent infection. The analysis detailed in this study will aid in the development of model systems to screen emergent coronaviruses as well as treatments to counteract infections.


Asunto(s)
/química , Betacoronavirus/fisiología , Replicación Viral , Secuencia de Aminoácidos , Animales , Betacoronavirus/metabolismo , Sitios de Unión , Línea Celular , Infecciones por Coronavirus/virología , Especificidad del Huésped , Humanos , Ratones , Modelos Moleculares , Mutación , Unión Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , /fisiología , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/metabolismo
17.
Nat Commun ; 12(1): 1715, 2021 03 17.
Artículo en Inglés | MEDLINE | ID: mdl-33731724

RESUMEN

The coronavirus spike glycoprotein, located on the virion surface, is the key mediator of cell entry and the focus for development of protective antibodies and vaccines. Structural studies show exposed sites on the spike trimer that might be targeted by antibodies with cross-species specificity. Here we isolated two human monoclonal antibodies from immunized humanized mice that display a remarkable cross-reactivity against distinct spike proteins of betacoronaviruses including SARS-CoV, SARS-CoV-2, MERS-CoV and the endemic human coronavirus HCoV-OC43. Both cross-reactive antibodies target the stem helix in the spike S2 fusion subunit which, in the prefusion conformation of trimeric spike, forms a surface exposed membrane-proximal helical bundle. Both antibodies block MERS-CoV infection in cells and provide protection to mice from lethal MERS-CoV challenge in prophylactic and/or therapeutic models. Our work highlights an immunogenic and vulnerable site on the betacoronavirus spike protein enabling elicitation of antibodies with unusual binding breadth.


Asunto(s)
Anticuerpos Monoclonales Humanizados/inmunología , Betacoronavirus/inmunología , Epítopos/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Animales , Anticuerpos Monoclonales Humanizados/uso terapéutico , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/uso terapéutico , Anticuerpos Antivirales/inmunología , Betacoronavirus/clasificación , Camelus , Infecciones por Coronavirus/tratamiento farmacológico , Infecciones por Coronavirus/virología , Reacciones Cruzadas , Epítopos/química , Epítopos/genética , Humanos , Ratones , Conformación Proteica , Subunidades de Proteína , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/genética
18.
Sci Adv ; 7(12)2021 03.
Artículo en Inglés | MEDLINE | ID: mdl-33741598

RESUMEN

Vaccination against SARS-CoV-2 provides an effective tool to combat the COVID-19 pandemic. Here, we combined antigen optimization and nanoparticle display to develop vaccine candidates for SARS-CoV-2. We first displayed the receptor-binding domain (RBD) on three self-assembling protein nanoparticle (SApNP) platforms using the SpyTag/SpyCatcher system. We then identified heptad repeat 2 (HR2) in S2 as the cause of spike metastability, designed an HR2-deleted glycine-capped spike (S2GΔHR2), and displayed S2GΔHR2 on SApNPs. An antibody column specific for the RBD enabled tag-free vaccine purification. In mice, the 24-meric RBD-ferritin SApNP elicited a more potent neutralizing antibody (NAb) response than the RBD alone and the spike with two stabilizing proline mutations in S2 (S2P). S2GΔHR2 elicited twofold higher NAb titers than S2P, while S2GΔHR2 SApNPs derived from multilayered E2p and I3-01v9 60-mers elicited up to 10-fold higher NAb titers. The S2GΔHR2-presenting I3-01v9 SApNP also induced critically needed T cell immunity, thereby providing a promising vaccine candidate.


Asunto(s)
/inmunología , Nanopartículas , Glicoproteína de la Espiga del Coronavirus , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , /química , /farmacología , Células HEK293 , Humanos , Inmunogenicidad Vacunal , Ratones , Ratones Endogámicos BALB C , Nanopartículas/química , Nanopartículas/uso terapéutico , Dominios Proteicos , /inmunología , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/inmunología , Glicoproteína de la Espiga del Coronavirus/farmacología
19.
Biointerphases ; 16(1): 011006, 2021 01 28.
Artículo en Inglés | MEDLINE | ID: mdl-33706521

RESUMEN

The novel coronavirus caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has reached more than 160 countries and has been declared a pandemic. SARS-CoV-2 infects host cells by binding to the angiotensin-converting enzyme 2 (ACE-2) surface receptor via the spike (S) receptor-binding protein (RBD) on the virus envelope. Global data on a similar infectious disease spread by SARS-CoV-1 in 2002 indicated improved stability of the virus at lower temperatures facilitating its high transmission in the community during colder months (December-February). Seasonal viral transmissions are strongly modulated by temperatures, which can impact viral trafficking into host cells; however, an experimental study of temperature-dependent activity of SARS-CoV-2 is still lacking. We mimicked SARS-CoV-2 with polymer beads coated with the SARS-CoV-2 S protein to study the effect of seasonal temperatures on the binding of virus-mimicking nanospheres to lung epithelia. The presence of the S protein RBD on nanosphere surfaces led to binding by Calu-3 airway epithelial cells via the ACE-2 receptor. Calu-3 and control fibroblast cells with S-RBD-coated nanospheres were incubated at 33 and 37 °C to mimic temperature fluctuations in the host respiratory tract, and we found no temperature dependence in contrast to nonspecific binding of bovine serum ablumin-coated nanospheres. Moreover, the ambient temperature changes from 4 to 40 °C had no effect on S-RBD-ACE-2 ligand-receptor binding and minimal effect on the S-RBD protein structure (up to 40 °C), though protein denaturing occurred at 51 °C. Our results suggest that ambient temperatures from 4 to 40 °C have little effect on the SARS-CoV-2-ACE-2 interaction in agreement with the infection data currently reported.


Asunto(s)
/metabolismo , Materiales Biocompatibles Revestidos , Células Epiteliales/metabolismo , Pulmón/metabolismo , Nanosferas , Glicoproteína de la Espiga del Coronavirus , Temperatura , /metabolismo , Animales , Línea Celular Tumoral , Materiales Biocompatibles Revestidos/química , Materiales Biocompatibles Revestidos/metabolismo , Células Epiteliales/patología , Células Epiteliales/virología , Humanos , Pulmón/patología , Pulmón/virología , Ratones , Células 3T3 NIH , /metabolismo , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/metabolismo
20.
Int J Mol Sci ; 22(5)2021 Feb 25.
Artículo en Inglés | MEDLINE | ID: mdl-33668756

RESUMEN

Despite sequence similarity to SARS-CoV-1, SARS-CoV-2 has demonstrated greater widespread virulence and unique challenges to researchers aiming to study its pathogenicity in humans. The interaction of the viral receptor binding domain (RBD) with its main host cell receptor, angiotensin-converting enzyme 2 (ACE2), has emerged as a critical focal point for the development of anti-viral therapeutics and vaccines. In this study, we selectively identify and characterize the impact of mutating certain amino acid residues in the RBD of SARS-CoV-2 and in ACE2, by utilizing our recently developed NanoBiT technology-based biosensor as well as pseudotyped-virus infectivity assays. Specifically, we examine the mutational effects on RBD-ACE2 binding ability, efficacy of competitive inhibitors, as well as neutralizing antibody activity. We also look at the implications the mutations may have on virus transmissibility, host susceptibility, and the virus transmission path to humans. These critical determinants of virus-host interactions may provide more effective targets for ongoing vaccines, drug development, and potentially pave the way for determining the genetic variation underlying disease severity.


Asunto(s)
/química , /metabolismo , /metabolismo , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/metabolismo , Secuencia de Aminoácidos , Anticuerpos Neutralizantes/inmunología , Antivirales/farmacología , Sitios de Unión , /inmunología , Células HEK293 , Interacciones Microbiota-Huesped , Humanos , Modelos Moleculares , Mutación , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Receptores Virales/química , Receptores Virales/metabolismo , Alineación de Secuencia
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