RESUMO
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.
Assuntos
Bacteriófagos , Bacteriófagos/metabolismo , DNA/metabolismoRESUMO
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.
RESUMO
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.
Assuntos
Vacinas contra COVID-19 , Polissacarídeos , Glicoproteína da Espícula de Coronavírus , Animais , Anticorpos Neutralizantes , Anticorpos Antivirais , COVID-19/prevenção & controle , Vacinas contra COVID-19/imunologia , Vacinas contra COVID-19/metabolismo , Humanos , Camundongos , Modelos Animais , SARS-CoV-2 , VacinaçãoRESUMO
[This corrects the article DOI: 10.1371/journal.ppat.1009352.].
RESUMO
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.
Assuntos
Anticorpos Monoclonais/farmacologia , Anticorpos Neutralizantes/farmacologia , Citotoxicidade Celular Dependente de Anticorpos , Vírus da Influenza A/imunologia , Células Matadoras Naturais/imunologia , Macrófagos Alveolares/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/farmacologia , Feminino , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/virologia , Macrófagos Alveolares/efeitos dos fármacos , Macrófagos Alveolares/virologia , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/virologiaRESUMO
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.
Assuntos
Anticorpos Antivirais/imunologia , COVID-19/imunologia , SARS-CoV-2/imunologia , Adulto , Enzima de Conversão de Angiotensina 2/antagonistas & inibidores , Enzima de Conversão de Angiotensina 2/imunologia , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/sangue , Anticorpos Antivirais/genética , Células Produtoras de Anticorpos/imunologia , Sítios de Ligação , Epitopos , Humanos , Imunoglobulina G/imunologia , Nucleocapsídeo/imunologia , Glicoproteína da Espícula de Coronavírus/imunologiaRESUMO
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.
Assuntos
Antivirais/uso terapêutico , Infecções por Coronavirus/tratamento farmacológico , Fabaceae/química , Infecções por Orthomyxoviridae/tratamento farmacológico , Lectinas de Plantas/uso terapêutico , Pneumonia Viral/tratamento farmacológico , Células A549 , Administração Intranasal , Animais , Antivirais/administração & dosagem , Antivirais/farmacologia , Betacoronavirus/efeitos dos fármacos , COVID-19 , Embrião de Galinha , Chlorocebus aethiops , Cães , Feminino , Humanos , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Células Madin Darby de Rim Canino , Camundongos , Camundongos Endogâmicos BALB C , Pandemias , Lectinas de Plantas/administração & dosagem , Lectinas de Plantas/farmacologia , Ligação Proteica , SARS-CoV-2 , Células Vero , Proteínas do Envelope Viral/metabolismoRESUMO
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.
Assuntos
Anticorpos Antivirais/química , Betacoronavirus/química , Infecções por Coronavirus/imunologia , Pneumonia Viral/imunologia , Glicoproteína da Espícula de Coronavírus/química , Adulto , Enzima de Conversão de Angiotensina 2 , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/metabolismo , Betacoronavirus/imunologia , Betacoronavirus/metabolismo , Sítios de Ligação , COVID-19 , Chlorocebus aethiops , Reações Cruzadas , Microscopia Crioeletrônica , Cristalografia por Raios X , Epitopos , Humanos , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/metabolismo , Masculino , Pandemias , Peptidil Dipeptidase A/metabolismo , Conformação Proteica , Domínios Proteicos , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Células VeroRESUMO
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.
Assuntos
Proteção Cruzada/imunologia , Ovos , Vacinas contra Influenza/administração & dosagem , Vacinas contra Influenza/imunologia , Influenza Humana/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Vacinação , Animais , Galinhas/anormalidades , Feminino , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Hemaglutininas/imunologia , Humanos , Vírus da Influenza A Subtipo H1N1/imunologia , Influenza Humana/prevenção & controle , Injeções Intramusculares , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidase/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Orthomyxoviridae/imunologia , Vacinas de Produtos Inativados/administração & dosagem , Vacinas de Produtos Inativados/imunologiaRESUMO
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.
Assuntos
Antineoplásicos/administração & dosagem , Cetuximab/administração & dosagem , Mitose/efeitos dos fármacos , Nanodiamantes , Paclitaxel/administração & dosagem , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cetuximab/química , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Modelos Animais de Doenças , Portadores de Fármacos , Sistemas de Liberação de Medicamentos , Receptores ErbB/genética , Receptores ErbB/metabolismo , Expressão Gênica , Humanos , Nanodiamantes/química , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Neoplasias/patologia , Paclitaxel/química , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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.