RESUMEN
The T5 family of viruses are tailed bacteriophages characterized by a long non-contractile tail. The bacteriophage DT57C is closely related to the paradigmal T5 phage, though it recognizes a different receptor (BtuB) and features highly divergent lateral tail fibers (LTF). Considerable portions of T5-like phages remain structurally uncharacterized. Here, we present the structure of DT57C determined by cryo-EM, and an atomic model of the virus, which was further explored using all-atom molecular dynamics simulations. The structure revealed a unique way of LTF attachment assisted by a dodecameric collar protein LtfC, and an unusual composition of the phage neck constructed of three protein rings. The tape measure protein (TMP) is organized within the tail tube in a three-stranded parallel α-helical coiled coil which makes direct contact with the genomic DNA. The presence of the C-terminal fragment of the TMP that remains within the tail tip suggests that the tail tip complex returns to its original state after DNA ejection. Our results provide a complete atomic structure of a T5-like phage, provide insights into the process of DNA ejection as well as a structural basis for the design of engineered phages and future mechanistic studies.
Asunto(s)
Bacteriófagos , Bacteriófagos/metabolismo , ADN/metabolismoRESUMEN
As the Internet of Things (IOT) becomes more widely used in our everyday lives, an increasing number of wireless communication devices are required, meaning that an increasing number of signals are transmitted and received through antennas. Thus, the performance of antennas plays an important role in IOT applications, and increasing the efficiency of antenna design has become a crucial topic. Antenna designers have often optimized antennas by using an EM simulation tool. Although this method is feasible, a great deal of time is often spent on designing the antenna. To improve the efficiency of antenna optimization, this paper proposes a design of experiments (DOE) method for antenna optimization. The antenna length and area in each direction were the experimental parameters, and the response variables were antenna gain and return loss. Response surface methodology was used to obtain optimal parameters for the layout of the antenna. Finally, we utilized antenna simulation software to verify the optimal parameters for antenna optimization, showing how the DOE method can increase the efficiency of antenna optimization. The antenna optimized by DOE was implemented, and its measured results show that the antenna gain and return loss were 2.65 dBi and 11.2 dB, respectively.
RESUMEN
A major challenge to end the pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is to develop a broadly protective vaccine that elicits long-term immunity. As the key immunogen, the viral surface spike (S) protein is frequently mutated, and conserved epitopes are shielded by glycans. Here, we revealed that S protein glycosylation has site-differential effects on viral infectivity. We found that S protein generated by lung epithelial cells has glycoforms associated with increased infectivity. Compared to the fully glycosylated S protein, immunization of S protein with N-glycans trimmed to the mono-GlcNAc-decorated state (SMG) elicited stronger immune responses and better protection for human angiotensin-converting enzyme 2 (hACE2) transgenic mice against variants of concern (VOCs). In addition, a broadly neutralizing monoclonal antibody was identified from SMG-immunized mice that could neutralize wild-type SARS-CoV-2 and VOCs with subpicomolar potency. Together, these results demonstrate that removal of glycan shields to better expose the conserved sequences has the potential to be an effective and simple approach for developing a broadly protective SARS-CoV-2 vaccine.
Asunto(s)
Vacunas contra la COVID-19 , Polisacáridos , Glicoproteína de la Espiga del Coronavirus , Animales , Anticuerpos Neutralizantes , Anticuerpos Antivirales , COVID-19/prevención & control , Vacunas contra la COVID-19/inmunología , Vacunas contra la COVID-19/metabolismo , Humanos , Ratones , Modelos Animales , SARS-CoV-2 , VacunaciónRESUMEN
[This corrects the article DOI: 10.1371/journal.ppat.1009352.].
RESUMEN
Hemagglutinin (HA) is the immunodominant protein of the influenza virus. We previously showed that mice injected with a monoglycosylated influenza A HA (HAmg) produced cross-strain-reactive antibodies and were better protected than mice injected with a fully glycosylated HA (HAfg) during lethal dose challenge. We employed a single B-cell screening platform to isolate the cross-protective monoclonal antibody (mAb) 651 from mice immunized with the HAmg of A/Brisbane/59/2007 (H1N1) influenza virus (Bris/07). The mAb 651 recognized the head domain of a broad spectrum of HAs from groups 1 and 2 influenza A viruses and offered prophylactic and therapeutic efficacy against A/California/07/2009 (H1N1) (Cal/09) and Bris/07 infections in mice. The antibody did not possess neutralizing activity; however, antibody-dependent cellular cytotoxicity and antibody-dependent cellular phagocytosis mediated by natural killer cells and alveolar macrophages were important in the protective efficacy of mAb 651. Together, this study highlighted the significance of effector functions for non-neutralizing antibodies to exhibit protection against influenza virus infection.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Anticuerpos Neutralizantes/farmacología , Citotoxicidad Celular Dependiente de Anticuerpos , Virus de la Influenza A/inmunología , Células Asesinas Naturales/inmunología , Macrófagos Alveolares/inmunología , Infecciones por Orthomyxoviridae/prevención & control , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/farmacología , Femenino , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/virología , Macrófagos Alveolares/efectos de los fármacos , Macrófagos Alveolares/virología , Ratones , Ratones Endogámicos BALB C , Infecciones por Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/virologíaRESUMEN
Serological and plasmablast responses and plasmablast-derived IgG monoclonal antibodies (MAbs) have been analysed in three COVID-19 patients with different clinical severities. Potent humoral responses were detected within 3 weeks of onset of illness in all patients and the serological titre was elicited soon after or concomitantly with peripheral plasmablast response. An average of 13.7% and 3.5% of plasmablast-derived MAbs were reactive with virus spike glycoprotein or nucleocapsid, respectively. A subset of anti-spike (10 of 32) antibodies cross-reacted with other betacoronaviruses tested and harboured extensive somatic mutations, indicative of an expansion of memory B cells upon SARS-CoV-2 infection. Fourteen of 32 anti-spike MAbs, including five anti-receptor-binding domain (RBD), three anti-non-RBD S1 and six anti-S2, neutralised wild-type SARS-CoV-2 in independent assays. Anti-RBD MAbs were further grouped into four cross-inhibiting clusters, of which six antibodies from three separate clusters blocked the binding of RBD to ACE2 and five were neutralising. All ACE2-blocking anti-RBD antibodies were isolated from two recovered patients with prolonged fever, which is compatible with substantial ACE2-blocking response in their sera. Finally, the identification of non-competing pairs of neutralising antibodies would offer potential templates for the development of prophylactic and therapeutic agents against SARS-CoV-2.
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Anticuerpos Antivirales/inmunología , COVID-19/inmunología , SARS-CoV-2/inmunología , Adulto , Enzima Convertidora de Angiotensina 2/antagonistas & inhibidores , Enzima Convertidora de Angiotensina 2/inmunología , Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/genética , Células Productoras de Anticuerpos/inmunología , Sitios de Unión , Epítopos , Humanos , Inmunoglobulina G/inmunología , Nucleocápside/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunologíaRESUMEN
The influenza virus hemagglutinin (HA) and coronavirus spike (S) protein mediate virus entry. HA and S proteins are heavily glycosylated, making them potential targets for carbohydrate binding agents such as lectins. Here, we show that the lectin FRIL, isolated from hyacinth beans (Lablab purpureus), has anti-influenza and anti-SARS-CoV-2 activity. FRIL can neutralize 11 representative human and avian influenza strains at low nanomolar concentrations, and intranasal administration of FRIL is protective against lethal H1N1 infection in mice. FRIL binds preferentially to complex-type N-glycans and neutralizes viruses that possess complex-type N-glycans on their envelopes. As a homotetramer, FRIL is capable of aggregating influenza particles through multivalent binding and trapping influenza virions in cytoplasmic late endosomes, preventing their nuclear entry. Remarkably, FRIL also effectively neutralizes SARS-CoV-2, preventing viral protein production and cytopathic effect in host cells. These findings suggest a potential application of FRIL for the prevention and/or treatment of influenza and COVID-19.
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Antivirales/uso terapéutico , Infecciones por Coronavirus/tratamiento farmacológico , Fabaceae/química , Infecciones por Orthomyxoviridae/tratamiento farmacológico , Lectinas de Plantas/uso terapéutico , Neumonía Viral/tratamiento farmacológico , Células A549 , Administración Intranasal , Animales , Antivirales/administración & dosificación , Antivirales/farmacología , Betacoronavirus/efectos de los fármacos , COVID-19 , Embrión de Pollo , Chlorocebus aethiops , Perros , Femenino , Humanos , Subtipo H1N1 del Virus de la Influenza A/efectos de los fármacos , Células de Riñón Canino Madin Darby , Ratones , Ratones Endogámicos BALB C , Pandemias , Lectinas de Plantas/administración & dosificación , Lectinas de Plantas/farmacología , Unión Proteica , SARS-CoV-2 , Células Vero , Proteínas del Envoltorio Viral/metabolismoRESUMEN
The COVID-19 pandemic has had an unprecedented health and economic impact and there are currently no approved therapies. We have isolated an antibody, EY6A, from an individual convalescing from COVID-19 and have shown that it neutralizes SARS-CoV-2 and cross-reacts with SARS-CoV-1. EY6A Fab binds the receptor binding domain (RBD) of the viral spike glycoprotein tightly (KD of 2 nM), and a 2.6-Å-resolution crystal structure of an RBD-EY6A Fab complex identifies the highly conserved epitope, away from the ACE2 receptor binding site. Residues within this footprint are key to stabilizing the pre-fusion spike. Cryo-EM analyses of the pre-fusion spike incubated with EY6A Fab reveal a complex of the intact spike trimer with three Fabs bound and two further multimeric forms comprising the destabilized spike attached to Fab. EY6A binds what is probably a major neutralizing epitope, making it a candidate therapeutic for COVID-19.
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Anticuerpos Antivirales/química , Betacoronavirus/química , Infecciones por Coronavirus/inmunología , Neumonía Viral/inmunología , Glicoproteína de la Espiga del Coronavirus/química , Adulto , Enzima Convertidora de Angiotensina 2 , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/metabolismo , Betacoronavirus/inmunología , Betacoronavirus/metabolismo , Sitios de Unión , COVID-19 , Chlorocebus aethiops , Reacciones Cruzadas , Microscopía por Crioelectrón , Cristalografía por Rayos X , Epítopos , Humanos , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/metabolismo , Masculino , Pandemias , Peptidil-Dipeptidasa A/metabolismo , Conformación Proteica , Dominios Proteicos , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus/inmunología , Glicoproteína de la Espiga del Coronavirus/metabolismo , Células VeroRESUMEN
Each year influenza virus infections cause hundreds of thousands of deaths worldwide and a significant level of morbidity with major economic burden. At the present time, vaccination with inactivated virus vaccine produced from embryonated chicken eggs is the most prevalent method to prevent the infections. However, current influenza vaccines are only effective against closely matched circulating strains and must be updated and administered yearly. Therefore, generating a vaccine that can provide broad protection is greatly needed for influenza vaccine development. We have previously shown that vaccination of the major surface glycoprotein hemagglutinin (HA) of influenza virus with a single N-acetylglucosamine at each of the N-glycosylation sites [monoglycosylated HA (HAmg)] can elicit better cross-protection compared with the fully glycosylated HA (HAfg). In the current study, we produced monoglycosylated inactivated split H1N1 virus vaccine from chicken eggs by the N-glycosylation process inhibitor kifunensine and the endoglycosidase Endo H, and intramuscularly immunized mice to examine its efficacy. Compared with vaccination of the traditional influenza vaccine with complex glycosylations from eggs, the monoglycosylated split virus vaccine provided better cross-strain protection against a lethal dose of virus challenge in mice. The enhanced antibody responses induced by the monoglycosylated vaccine immunization include higher neutralization activity, higher hemagglutination inhibition, and more HA stem selectivity, as well as, interestingly, higher antibody-dependent cellular cytotoxicity. This study provides a simple and practical procedure to enhance the cross-strain protection of influenza vaccine by removing the outer part of glycans from the virus surface through modifications of the current egg-based process.
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Protección Cruzada/inmunología , Huevos , Vacunas contra la Influenza/administración & dosificación , Vacunas contra la Influenza/inmunología , Gripe Humana/inmunología , Infecciones por Orthomyxoviridae/prevención & control , Vacunación , Animales , Pollos/anomalías , Femenino , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Hemaglutininas/inmunología , Humanos , Subtipo H1N1 del Virus de la Influenza A/inmunología , Gripe Humana/prevención & control , Inyecciones Intramusculares , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidasa/inmunología , Ratones , Ratones Endogámicos BALB C , Infecciones por Orthomyxoviridae/inmunología , Vacunas de Productos Inactivados/administración & dosificación , Vacunas de Productos Inactivados/inmunologíaRESUMEN
The poor intracellular uptake and non-specific binding of anticancer drugs into cancer cells are the bottlenecks in cancer therapy. Nanocarrier platforms provide the opportunities to improve the drug efficacy. Here we show a carbon-based nanomaterial nanodiamond (ND) that carried paclitaxel (PTX), a microtubule inhibitor, and cetuximab (Cet), a specific monoclonal antibody against epidermal growth factor receptor (EGFR), inducing mitotic catastrophe and tumor inhibition in human colorectal cancer (CRC). ND-PTX blocked the mitotic progression, chromosomal separation, and induced apoptosis in the CRC cells; however, NDs did not induce these effects. Conjugation of ND-PTX with Cet (ND-PTX-Cet) was specifically binding to the EGFR-positive CRC cells and enhanced the mitotic catastrophe and apoptosis induction. Besides, ND-PTX-Cet markedly decreased tumor size in the xenograft EGFR-expressed human CRC tumors of nude mice. Moreover, ND-PTX-Cet induced the mitotic marker protein phospho-histone 3 (Ser10) and apoptotic protein active-caspase 3 for mitotic catastrophe and apoptosis. Taken together, this study demonstrated that the co-delivery of PTX and Cet by ND enhanced the effects of mitotic catastrophe and apoptosis in vitro and in vivo, which may be applied in the human CRC therapy.
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Antineoplásicos/administración & dosificación , Cetuximab/administración & dosificación , Mitosis/efectos de los fármacos , Nanodiamantes , Paclitaxel/administración & dosificación , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Cetuximab/química , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Modelos Animales de Enfermedad , Portadores de Fármacos , Sistemas de Liberación de Medicamentos , Receptores ErbB/genética , Receptores ErbB/metabolismo , Expresión Génica , Humanos , Nanodiamantes/química , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Neoplasias/patología , Paclitaxel/química , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Selective macroautophagy/autophagy plays a pivotal role in the processing of foreign pathogens and cellular components to maintain homeostasis in human cells. To date, numerous studies have demonstrated the uptake of nanoparticles by cells, but their intracellular processing through selective autophagy remains unclear. Here we show that carbon-based nanodiamonds (NDs) coated with ubiquitin (Ub) bind to autophagy receptors (SQSTM1 [sequestosome 1], OPTN [optineurin], and CALCOCO2/NDP52 [calcium binding and coiled-coil domain 2]) and are then linked to MAP1LC3/LC3 (microtubule-associated protein 1 light chain 3) for entry into the selective autophagy pathway. NDs are ultimately delivered to lysosomes. Ectopically expressed SQSTM1-green fluorescence protein (GFP) could bind to the Ub-coated NDs. By contrast, the Ub-associated domain mutant of SQSTM1 (ΔUBA)-GFP did not bind to the Ub-coated NDs. Chloroquine, an autophagy inhibitor, prevented the ND-containing autophagosomes from fusing with lysosomes. Furthermore, autophagy receptors OPTN and CALCOCO2/NDP52, involved in the processing of bacteria, were found to be involved in the selective autophagy of NDs. However, ND particles located in the lysosomes of cells did not induce mitotic blockage, senescence, or cell death. Single ND clusters in the lysosomes of cells were observed in the xenografted human lung tumors of nude mice. This study demonstrated for the first time that Ub-coated nanoparticles bind to autophagy receptors for entry into the selective autophagy pathway, facilitating their delivery to lysosomes.