RESUMEN
The ongoing Coronavirus Disease 2019 (COVID-19) pandemic caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) threatens global public health and economy unprecedentedly, requiring accelerating development of prophylactic and therapeutic interventions. Molecular understanding of neutralizing antibodies (NAbs) would greatly help advance the development of monoclonal antibody (mAb) therapy, as well as the design of next generation recombinant vaccines. Here, we applied H2L2 transgenic mice encoding the human immunoglobulin variable regions, together with a state-of-the-art antibody discovery platform to immunize and isolate NAbs. From a large panel of isolated antibodies, 25 antibodies showed potent neutralizing activities at sub-nanomolar levels by engaging the spike receptor-binding domain (RBD). Importantly, one human NAb, termed PR1077, from the H2L2 platform and 2 humanized NAb, including PR953 and PR961, were further characterized and subjected for subsequent structural analysis. High-resolution X-ray crystallography structures unveiled novel epitopes on the receptor-binding motif (RBM) for PR1077 and PR953, which directly compete with human angiotensin-converting enzyme 2 (hACE2) for binding, and a novel non-blocking epitope on the neighboring site near RBM for PR961. Moreover, we further tested the antiviral efficiency of PR1077 in the Ad5-hACE2 transduction mouse model of COVID-19. A single injection provided potent protection against SARS-CoV-2 infection in either prophylactic or treatment groups. Taken together, these results shed light on the development of mAb-related therapeutic interventions for COVID-19.
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Anticuerpos Neutralizantes/inmunología , COVID-19/virología , SARS-CoV-2/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/metabolismo , Anticuerpos Neutralizantes/metabolismo , Anticuerpos Neutralizantes/ultraestructura , Anticuerpos Antivirales/inmunología , COVID-19/epidemiología , COVID-19/inmunología , COVID-19/metabolismo , Epítopos/inmunología , Humanos , Ratones , Ratones Transgénicos , Pruebas de Neutralización , Pandemias , Unión Proteica , Dominios Proteicos , Receptores Virales/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunologíaRESUMEN
The rapid emergence and spread of escaping mutations of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has significantly challenged our efforts in fighting against the COVID-19 pandemic. A broadly neutralizing reagent against these concerning variants is thus highly desirable for the prophylactic and therapeutic treatments of SARS-CoV-2 infection. We herein report a covalent engineering strategy on protein minibinders for potent neutralization of the escaping variants such as B.1.617.2 (Delta), B.1.617.1 (Kappa), and B.1.1.529 (Omicron) through in situ cross-linking with the spike receptor binding domain (RBD). The resulting covalent minibinder (GlueBinder) exhibited enhanced blockage of RBD-human angiotensin-converting enzyme 2 (huACE2) interaction and more potent neutralization effect against the Delta variant than its noncovalent counterpart as demonstrated on authentic virus. By leveraging the covalent chemistry against escaping mutations, our strategy may be generally applicable for restoring and enhancing the potency of neutralizing antibodies to SARS-CoV-2 and other rapidly evolving viral targets.
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Tratamiento Farmacológico de COVID-19 , SARS-CoV-2 , Anticuerpos Neutralizantes , Anticuerpos Antivirales , Humanos , Pruebas de Neutralización , Pandemias , Unión Proteica , SARS-CoV-2/genética , Glicoproteína de la Espiga del Coronavirus/genéticaRESUMEN
BACKGROUND: Genomic testing of somatic and germline DNA has transformed cancer care. However, low genetic knowledge among patients may compromise care and health outcomes. Given the rise in genomic testing, we sought to understand patients' knowledge of their genetic test results. MATERIALS AND METHODS: We conducted a survey-based study with 85 patients at a comprehensive cancer center. We compared self-reported recall of (a) having had somatic/germline testing and (b) their specific somatic/germline results to the genomic test results documented in the medical record. RESULTS: Approximately 30% of patients did not recall having had testing. Of those who recalled having testing, 44% of patients with pathogenic/likely pathogenic germline mutations and 57% of patients with reported somatic alterations did not accurately recall their specific gene or variant-level results. CONCLUSION: Given significant knowledge gaps in patients' recall of genomic testing, there is a critical need to improve patient-directed education and return-of-results strategies.
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Genómica , Neoplasias , Perfil Genético , Pruebas Genéticas , Mutación de Línea Germinal , Humanos , Neoplasias/genéticaRESUMEN
Baculovirus entry into insect midgut cells is dependent on a multiprotein complex of per os infectivity factors (PIFs) on the envelopes of occlusion-derived virions (ODVs). The structure and assembly of the PIF complex are largely unknown. To reveal the complete members of the complex, a combination of blue native polyacrylamide gel electrophoresis, liquid chromatography-tandem mass spectrometry, and Western blotting was conducted on three different baculoviruses. The results showed that the PIF complex has a molecular mass of â¼500 kDa and consists of nine PIFs, including a newly discovered member (PIF9). To decipher the assembly process, each pif gene was knocked out from the Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) genome individually by use of synthetic baculovirus technology, and the impact on PIF complex formation was investigated. Deletion of pif8 resulted in the formation of an â¼400-kDa subcomplex. Deletion of pif0, -4, -6, -7, or -9 resulted in a subcomplex of â¼230 kDa, but deletion of pif1, -2, or -3 abolished formation of any complex. Taken together, our data identified a core complex of â¼230 kDa, consisting of PIF1, -2, and -3. This revised the previous knowledge that the core complex was about 170 kDa and contained PIF1 to -4. Analysis of the PIF complex in cellular fractions suggested that it is assembled in the cytoplasm before being transported to the nucleus and subsequently incorporated into the envelopes of ODVs. Only the full complex, not the subcomplex, is resistant to proteolytic attack, indicating the essentiality of correct complex assembly for oral infection.IMPORTANCE Entry of baculovirus into host insects is mediated by a per os infectivity factor (PIF) complex on the envelopes of occlusion-derived viruses (ODVs). Knowledge of the composition and structure of the PIF complex is fundamental to understanding its mode of action. By using multiple approaches, we determined the complete list of proteins (nine) in the PIF complex. In contrast to previous knowledge in the field, the core complex is revised to â¼230 kDa and consists of PIF1 to -3 but not PIF4. Interestingly, our results suggest that the PIF complex is formed in the cytoplasm prior to its transport to the nucleus and subsequent incorporation into ODVs. Only the full complex is resistant to proteolytic degradation in the insect midgut, implying the critical role of the entire complex. These findings provide the baseline for future studies on the ODV entry mechanism mediated by the multiprotein complex.
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Baculoviridae/metabolismo , Baculoviridae/patogenicidad , Factores de Virulencia/metabolismo , Animales , Línea Celular , Infecciones por Virus ADN , Insectos/virología , Nucleopoliedrovirus/patogenicidad , Células Sf9 , Proteínas del Envoltorio Viral/metabolismo , Virión/patogenicidadRESUMEN
BACKGROUND: Public health resources are limited and best used for effective programs. This study explores associations of mis-implementation in public health (ending effective programs or continuing ineffective programs) with organizational supports for evidence-based decision making among U.S. local health departments. METHODS: The national U.S. sample for this cross-sectional study was stratified by local health department jurisdiction population size. One person was invited from each randomly selected local health department: the leader in chronic disease, or the director. Of 600 selected, 579 had valid email addresses; 376 completed the survey (64.9% response). Survey items assessed frequency of and reasons for mis-implementation. Participants indicated agreement with statements on organizational supports for evidence-based decision making (7-point Likert). RESULTS: Thirty percent (30.0%) reported programs often or always ended that should have continued (inappropriate termination); organizational supports for evidence-based decision making were not associated with the frequency of programs ending. The main reason given for inappropriate termination was grant funding ended (86.0%). Fewer (16.4%) reported programs often or always continued that should have ended (inappropriate continuation). Higher perceived organizational supports for evidence-based decision making were associated with less frequent inappropriate continuation (odds ratio = 0.86, 95% confidence interval 0.79, 0.94). All organizational support factors were negatively associated with inappropriate continuation. Top reasons were sustained funding (55.6%) and support from policymakers (34.0%). CONCLUSIONS: Organizational supports for evidence-based decision making may help local health departments avoid continuing programs that should end. Creative mechanisms of support are needed to avoid inappropriate termination. Understanding what influences mis-implementation can help identify supports for de-implementation of ineffective programs so resources can go towards evidence-based programs.
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Práctica Clínica Basada en la Evidencia , Evaluación de Programas y Proyectos de Salud , Administración en Salud Pública , Enfermedad Crónica , Estudios Transversales , Toma de Decisiones , Femenino , Humanos , Liderazgo , Gobierno Local , Masculino , Oportunidad Relativa , Asignación de Recursos , Encuestas y Cuestionarios , Estados UnidosRESUMEN
OBJECTIVES: To determine the extent to which US local health departments (LHDs) are engaged in evidence-based public health and whether this is influenced by the presence of an academic health department (AHD) partnership. METHODS: We surveyed a cross-sectional stratified random sample of 579 LHDs in 2017. We ascertained the extent of support for evidence-based decision-making and the use of evidence-based interventions in several chronic disease programs and whether the LHD participated in a formal, informal, or no AHD partnership. RESULTS: We received 376 valid responses (response rate 64.9%). There were 192 (51.6%) LHDs with a formal, 80 (21.6%) with an informal, and 99 (26.7%) with no AHD partnership. Participants with formal AHD partnerships reported higher perceived organizational supports for evidence-based decision-making and interventions compared with either informal or no AHD partnerships. The odds of providing 1 or more chronic disease evidence-based intervention were significantly higher in LHDs with formal AHD partnerships compared with LHDs with no AHD partnerships (adjusted odds ratio = 2.3; 95% confidence interval = 1.3, 4.0). CONCLUSIONS: Formal academic-practice partnerships can be important means for advancing evidence-based decision-making and for implementing evidence-based programs and policies.
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Planificación en Salud Comunitaria/organización & administración , Prevención Primaria/organización & administración , Asociación entre el Sector Público-Privado/organización & administración , Conducta Cooperativa , Estudios Transversales , Humanos , Gobierno Local , Salud PúblicaRESUMEN
OBJECTIVE: Use of research evidence in public health decision making can be affected by organizational supports. Study objectives are to identify patterns of organizational supports and explore associations with research evidence use for job tasks among public health practitioners. DESIGN: In this longitudinal study, we used latent class analysis to identify organizational support patterns, followed by mixed logistic regression analysis to quantify associations with research evidence use. SETTING: The setting included 12 state public health department chronic disease prevention units and their external partnering organizations involved in chronic disease prevention. PARTICIPANTS: Chronic disease prevention staff from 12 US state public health departments and partnering organizations completed self-report surveys at 2 time points, in 2014 and 2016 (N = 872). MAIN OUTCOME MEASURES: Latent class analysis was employed to identify subgroups of survey participants with distinct patterns of perceived organizational supports. Two classify-analyze approaches (maximum probability assignment and multiple pseudo-class draws) were used in 2017 to investigate the association between latent class membership and research evidence use. RESULTS: The optimal model identified 4 latent classes, labeled as "unsupportive workplace," "low agency leadership support," "high agency leadership support," and "supportive workplace." With maximum probability assignment, participants in "high agency leadership support" (odds ratio = 2.08; 95% CI, 1.35-3.23) and "supportive workplace" (odds ratio = 1.74; 95% CI, 1.10-2.74) were more likely to use research evidence in job tasks than "unsupportive workplace." The multiple pseudo-class draws produced comparable results with odds ratio = 2.09 (95% CI, 1.31-3.30) for "high agency leadership support" and odds ratio = 1.74 (95% CI, 1.07-2.82) for "supportive workplace." CONCLUSIONS: Findings suggest that leadership support may be a crucial element of organizational supports to encourage research evidence use. Organizational supports such as supervisory expectations, access to evidence, and participatory decision making may need leadership support as well to improve research evidence use in public health job tasks.
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Práctica de Salud Pública/normas , Investigación/normas , Gobierno Estatal , Adulto , Enfermedad Crónica/prevención & control , Femenino , Humanos , Análisis de Clases Latentes , Estudios Longitudinales , Masculino , Persona de Mediana Edad , Oportunidad Relativa , Práctica de Salud Pública/estadística & datos numéricos , Investigación/estadística & datos numéricosRESUMEN
The high risk of SARS-CoV-2 infection and reinfection and the occurrence of post-acute pulmonary sequelae have highlighted the importance of understanding the mechanism underlying lung repair after injury. To address this concern, comparative and systematic analyses of SARS-CoV-2 infection in COVID-19 patients and animals were conducted. In the lungs of nine patients who died of COVID-19 and one recovered from COVID-19 but died of unrelated disease in early 2020, damage-related transient progenitor (DATP) cells expressing CK8 marker proliferated significantly. These CK8+ DATP cells were derived from bronchial CK5+ basal cells. However, they showed different cell fate toward differentiation into type I alveolar cells in the deceased and convalescent patients, respectively. By using a self-limiting hamster infection model mimicking the dynamic process of lung injury remodeling in mild COVID-19 patients, the accumulation and regression of CK8+ cell marker were found to be closely associated with the disease course. Finally, we examined the autopsied lungs of two patients who died of infection by the recent Omicron variant and found that they only exhibited mild pathological injury with no CK8+ cell proliferation. These results indicate a clear pulmonary cell remodeling route and suggest that CK8+ DATP cells play a primary role in mediating alveolar remodeling, highlighting their potential applications as diagnostic markers and therapeutic targets.
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COVID-19 , SARS-CoV-2 , COVID-19/inmunología , COVID-19/patología , COVID-19/virología , Humanos , Animales , Masculino , Persona de Mediana Edad , Femenino , Cricetinae , Pulmón/patología , Pulmón/virología , Pulmón/inmunología , Adulto , Alveolos Pulmonares/patología , Alveolos Pulmonares/virología , Alveolos Pulmonares/inmunología , Anciano , Modelos Animales de Enfermedad , Lesión Pulmonar/virología , Lesión Pulmonar/patología , Lesión Pulmonar/inmunología , Proliferación Celular , Diferenciación CelularRESUMEN
The ongoing COVID-19 pandemic has caused over six million deaths and huge economic burdens worldwide. Antivirals against its causative agent, SARS-CoV-2, are in urgent demand. Previously, we reported that heterocylic compounds, i.e., chloroquine (CQ) and hydroxychloroquine (HCQ), are potent in inhibiting SARS-CoV-2 replication in vitro. In this study, we discussed the syntheses of two novel heterocylic compounds: tert-butyl rel-4-(((3R,4S)-3-(1H-indol-3-yl)-1-oxo-2-propyl-1,2,3,4-tetrahydroisoquinolin-4-yl)methyl)piperazine-1-carboxylate (trans-1) and rel-(3R,4S)-3-(1H-indol-3-yl)-4-(piperazin-1-ylmethyl)-2-propyl-3,4-dihydroisoquinolin-1(2H)-one (trans-2), which effectively suppressed authentic SARS-CoV-2 replication in Vero E6 cells. Compound trans-1 showed higher anti-SARS-CoV-2 activity than trans-2, with a half maximal effective concentration (EC50) of 3.15 µM and a selective index (SI) exceeding 63.49, which demonstrated comparable potency to CQ or HCQ. Additional anti-SARS-CoV-2 tests on Calu-3 human lung cells showed that trans-1 efficiently inhibited viral replication (EC50 = 2.78 µM; SI: > 71.94) and performed better than CQ (EC50 = 44.90 µM; SI = 2.94). The time of an addition assay showed that the action mechanism of trans-1 differed from that of CQ, as it mainly inhibited the post-entry viral replication in both Vero E6 and Calu-3 cells. In addition, the differences between the antiviral mechanisms of these novel compounds and CQ were discussed.
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COVID-19 , Compuestos Heterocíclicos , Tetrahidroisoquinolinas , Humanos , SARS-CoV-2 , Pandemias , Tetrahidroisoquinolinas/farmacología , Cloroquina/farmacología , Hidroxicloroquina/farmacología , Antivirales/farmacologíaRESUMEN
Herpes simplex virus type 1 (HSV-1) causes lifelong infections worldwide, and currently there is no efficient cure or vaccine. HSV-1-derived tools, such as neuronal circuit tracers and oncolytic viruses, have been used extensively; however, further genetic engineering of HSV-1 is hindered by its complex genome structure. In the present study, we designed and constructed a synthetic platform for HSV-1 based on H129-G4. The complete genome was constructed from 10 fragments through 3 rounds of synthesis using transformation-associated recombination (TAR) in yeast, and was named H129-Syn-G2. The H129-Syn-G2 genome contained two copies of the gfp gene and was transfected into cells to rescue the virus. According to growth curve assay and electron microscopy results, the synthetic viruses exhibited more optimized growth properties and similar morphogenesis compared to the parental virus. This synthetic platform will facilitate further manipulation of the HSV-1 genome for the development of neuronal circuit tracers, oncolytic viruses, and vaccines.
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Herpesvirus Humano 1 , Herpesvirus Humano 1/genética , NeuronasRESUMEN
BACKGROUND: The ongoing coronavirus disease 2019 (COVID-19) pandemic has had an enormous impact on our societies. Moreover, the disease's extensive and sustained symptoms are now becoming a nonnegligible medical challenge. In this respect, data indicate that heart failure is one of the most common readmission diagnoses among COVID-19 patients. METHODS: In this study, we used human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes to develop an in vitro model of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and studied the dynamic changes occurring in cardiomyocytes after SARS-CoV-2 infection. RESULTS: To this end, we have created an effective time series SARS-CoV-2 infection model exhibiting different functional patterns of up- and downregulated proteins, and demonstrating that SARS-CoV-2 mainly affects (i) the lipid and the energy metabolism of hiPSC-derived cardiomyocytes during the early infection stage, and (ii) the DNA repair ability of cardiomyocytes during the late infection stage. By analyzing the proteome changes occurring at different infection timepoints, we were able to observe that the simulated disease (COVID-19) course developed rapidly, and that each of the studied timepoints was characterized by a distinct protein expression pattern. CONCLUSIONS: Our findings highlight the importance of early detection and personalized treatment based on the disease stage. Finally, by combing the proteomics data with virus-host interaction network analysis, we were able to identify several potential drug targets for the disease.
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COVID-19 , Insuficiencia Cardíaca , Células Madre Pluripotentes Inducidas , Humanos , SARS-CoV-2 , Células Madre Pluripotentes Inducidas/metabolismo , Miocitos Cardíacos/metabolismo , Insuficiencia Cardíaca/metabolismoRESUMEN
Neutralizing monoclonal antibodies (mAb) are a major therapeutic strategy for the treatment of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. The continuous emergence of new SARS-CoV-2 variants worldwide has increased the urgency for the development of new mAbs. In this study, we immunized mice with the receptor-binding domain (RBD) of the SARS-CoV-2 prototypic strain (WIV04) and screened 35 RBD-specific mAbs using hybridoma technology. Results of the plaque reduction neutralization test showed that 25 of the mAbs neutralized authentic WIV04 strain infection. The 25 mAbs were divided into three categories based on the competitive enzyme-linked immunosorbent assay results. A representative mAb was selected from each category (RD4, RD10, and RD14) to determine the binding kinetics and median inhibitory concentration (IC50) of WIV04 and two variants of concern (VOC): B.1.351 (Beta) and B.1.617.2 (Delta). RD4 neutralized the B.1.617.2 variant with an IC50 of 2.67 âng/mL; however, it completely lost neutralizing activity against the B.1.351 variant. RD10 neutralized both variants with an IC50 exceeding 100 âng/mL; whereas RD14 neutralized two variants with a higher IC50 (>1 âmg/mL). Animal experiments were performed to evaluate the protective effects of RD4 and RD10 against various VOC infections. RD4 could protect Adv-hACE2 transduced mice from B.1.617.2 infection at an antibody concentration of 25 âmg/kg, while RD10 could protect mice from B.1.351 infection at an antibody concentration of 75 âmg/kg. These results highlight the potential for future modifications of the mAbs for practical use.
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Anticuerpos Monoclonales , COVID-19 , Animales , Humanos , Ratones , Hibridomas , SARS-CoV-2 , Anticuerpos Antivirales , Glicoproteína de la Espiga del Coronavirus , Anticuerpos Neutralizantes , Pruebas de NeutralizaciónRESUMEN
Tetrandrine (TET) has been used to treat silicosis in China for decades. The aim of this study was to facilitate rational repurposing of TET against SARS-CoV-2 infection. In this study, we confirmed that TET exhibited antiviral potency against SARS-CoV-2 in the African green monkey kidney (Vero E6), human hepatocarcinoma (Huh7), and human lung adenocarcinoma epithelial (Calu-3) cell lines. TET functioned during the early-entry stage of SARS-CoV-2 and impeded intracellular trafficking of the virus from early endosomes to endolysosomes. An in vivo study that used adenovirus (AdV) 5-human angiotensin-converting enzyme 2 (hACE2)-transduced mice showed that although TET did not reduce pulmonary viral load, it significantly alleviated pathological damage in SARS-CoV-2-infected murine lungs. The systemic preclinical pharmacokinetics were investigated based on in vivo and in vitro models, and the route-dependent biodistribution of TET was explored. TET had a large volume of distribution, which contributed to its high tissue accumulation. Inhaled administration helped TET target the lung and reduced its exposure to other tissues, which mitigated its off-target toxicity. Based on the available human pharmacokinetic data, it appeared feasible to achieve an unbound TET 90% maximal effective concentration (EC90) in human lungs. This study provides insights into the route-dependent pulmonary biodistribution of TET associated with its efficacy.
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The global emergence of SARS-CoV-2 variants has led to increasing breakthrough infections in vaccinated populations, calling for an urgent need to develop more effective and broad-spectrum vaccines to combat COVID-19. Here we report the preclinical development of RQ3013, an mRNA vaccine candidate intended to bring broad protection against SARS-CoV-2 variants of concern (VOCs). RQ3013, which contains pseudouridine-modified mRNAs formulated in lipid nanoparticles, encodes the spike (S) protein harboring a combination of mutations responsible for immune evasion of VOCs. Here we characterized the expressed S immunogen and evaluated the immunogenicity, efficacy, and safety of RQ3013 in various animal models. RQ3013 elicited robust immune responses in mice, hamsters, and nonhuman primates (NHP). It can induce high titers of antibodies with broad cross-neutralizing ability against the wild-type, B.1.1.7, B.1.351, B.1.617.2, and the newly emerging Omicron variants. In mice and NHP, two doses of RQ3013 protected the upper and lower respiratory tract against infection by SARS-CoV-2 and its variants. Furthermore, our safety assessment of RQ3013 in NHP showed no observable adverse effects. These results provide strong support for the evaluation of RQ3013 in clinical trials and suggest that it may be a promising candidate for broad protection against COVID-19 and its variants.
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Vacunas contra la COVID-19 , COVID-19 , Vacunas de ARNm , Animales , Cricetinae , Ratones , COVID-19/prevención & control , Vacunas contra la COVID-19/inmunología , Vacunas de ARNm/inmunología , SARS-CoV-2/genética , Primates , Inmunogenicidad Vacunal , Anticuerpos ampliamente neutralizantes , Anticuerpos AntiviralesRESUMEN
The engineering of viral genomes facilitates both fundamental and applied research on viruses. However, the multiloci manipulation of DNAs of viruses with large DNA genomes, such as baculoviruses, herpesviruses, and poxviruses, is technically challenging, particularly for highly homologous or repetitive sequences. Homologous regions (hrs) have multiple copies in many large DNA viruses and play pivotal roles in the viral life cycle. Here, we used synthetic biology to investigate the fundamental function of baculoviral hrs by conducting multiloci manipulation of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) DNA that contains eight hrs scattered in the genome. Using transformation-associated recombination in yeast, we generated recombinant AcMNPV genomes in which we deleted all hrs or retained a single hr (hr1, hr2, or hr3). Infectious viruses were rescued after transfecting the synthetic viral genomes into host cells, and their replication features were characterized. The results demonstrated that deletion of all hrs severely compromised viral DNA replication and progeny production, whereas retaining only a single hr was essential for efficient viral DNA replication and progeny production. The synthetic virus with hr2 or hr3 showed a growth curve similar to that of the parental virus. Transcriptomic analysis revealed that hr1, hr2, and hr3 could enhance gene transcription within a surrounding region of 14.6 kb, 13.8 kb, and 29.8 kb, respectively. Overall, this study revealed the advantages of synthetic biology in multiloci engineering and functional studies of large DNA viruses. In addition, our findings on hrs will be helpful for the design and improvement of baculovirus-based expression vectors.
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Baculoviridae , Replicación Viral , Animales , Baculoviridae/genética , Replicación del ADN/genética , ADN Viral/genética , Spodoptera/genética , Replicación Viral/genéticaRESUMEN
Baculoviruses have been widely used as expression vectors. However, numerous genes in the baculoviral genome are non-essential for cellular infection and protein expression, making the optimisation of baculovirus expression vectors possible. We used a synthetic biological method to reduce the number of genes in a partial region of the autograph californica multiple nucleopolyhedrovirus (AcMNPV), the most widely used baculovirus expression vector. The C1 region of the AcMNPV is 46.4 kb and is subdivided into B1, B2, and B3 fragments. We first designed modified B1, B2, and B3 fragments by deleting the non-essential genes, and then synthesised complete viral genomes containing either individual modified B fragments or joint modified B fragments through transformation-related recombination in yeast. The synthetic genomes were then transfected into Sf9 cells to rescue the progeny viruses and test their infectivity. The design-build-test cycle was repeated until the ultimately rescued virus could produce progeny viruses efficiently. Finally, AcMNPV-Syn-mC1-1.1 by deleting approximately 17.2 kb, including 20 ORFs, in the C1 region, was obtained. This is essential to the synthesis of a minimal AcMNPV genome that can generate infectious progeny viruses and can be further used to optimise the foundation of baculovirus expression vectors.
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Nucleopoliedrovirus , Animales , Nucleopoliedrovirus/genética , Spodoptera , Baculoviridae , Células Sf9 , Replicación Viral/genéticaRESUMEN
Coronavirus disease 2019 (COVID-19) patients with impact on skin and hair loss are reported. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is detected in the skin of some patients; however, the detailed pathological features of skin tissues from patients infected with SARS-CoV-2 at a molecular level are limited. Especially, the ability of SARS-CoV-2 to infect skin cells and impact their function is not well understood. A proteome map of COVID-19 skin is established here and the susceptibility of human-induced pluripotent stem cell (hiPSC)-derived skin organoids with hair follicles and nervous system is investigated, to SARS-CoV-2 infection. It is shown that KRT17+ hair follicles can be infected by SARS-CoV-2 and are associated with the impaired development of hair follicles and epidermis. Different types of nervous system cells are also found to be infected, which can lead to neuron death. Findings from the present work provide evidence for the association between COVID-19 and hair loss. hiPSC-derived skin organoids are also presented as an experimental model which can be used to investigate the susceptibility of skin cells to SARS-CoV-2 infection and can help identify various pathological mechanisms and drug screening strategies.
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COVID-19/fisiopatología , Células Madre Pluripotentes Inducidas/citología , Modelos Biológicos , Organoides/citología , Piel/citología , COVID-19/virología , Folículo Piloso/virología , Humanos , Sistema Nervioso/virología , Proteómica , SARS-CoV-2/aislamiento & purificaciónRESUMEN
Coronavirus disease 2019 (COVID-19) remains a global public health threat. Hence, more effective and specific antivirals are urgently needed. Here, COVID-19 hyperimmune globulin (COVID-HIG), a passive immunotherapy, is prepared from the plasma of healthy donors vaccinated with BBIBP-CorV (Sinopharm COVID-19 vaccine). COVID-HIG shows high-affinity binding to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein, the receptor-binding domain (RBD), the N-terminal domain of the S protein, and the nucleocapsid protein; and blocks RBD binding to human angiotensin-converting enzyme 2 (hACE2). Pseudotyped and authentic virus-based assays show that COVID-HIG displays broad-spectrum neutralization effects on a wide variety of SARS-CoV-2 variants, including D614G, Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Kappa (B.1.617.1), Delta (B.1.617.2), and Omicron (B.1.1.529) in vitro. However, a significant reduction in the neutralization titer is detected against Beta, Delta, and Omicron variants. Additionally, assessments of the prophylactic and treatment efficacy of COVID-HIG in an Adv5-hACE2-transduced IFNAR-/- mouse model of SARS-CoV-2 infection show significantly reduced weight loss, lung viral loads, and lung pathological injury. Moreover, COVID-HIG exhibits neutralization potency similar to that of anti-SARS-CoV-2 hyperimmune globulin from pooled convalescent plasma. Overall, the results demonstrate the potential of COVID-HIG against SARS-CoV-2 infection and provide reference for subsequent clinical trials.
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Vacunas contra la COVID-19 , COVID-19 , Globulinas , Animales , COVID-19/terapia , Globulinas/uso terapéutico , Humanos , Inmunización Pasiva , Ratones , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , Sueroterapia para COVID-19RESUMEN
Baculoviruses are large DNA viruses which have been widely used as expression vectors and biological insecticides. Homologous recombination and Bac-to-Bac system have been the main methods for manipulating the baculovirus genome. Recently, we generated a synthetic baculovirus AcMNPV-WIV-Syn1 which fully resembled its parental virus Autographa californica multiple nucleopolyhedrovirus (AcMNPV). Here, we report the modification of AcMNPV-WIV-Syn1 into a novel bacmid, AcBac-Syn, which can be used as a backbone for Bac-to-Bac system. To achieve this, a vector contained a LacZ:attTn7 and egfp cassette was constructed, and recombined with a linearized AcMNPV-WIV-Syn1 genome by transformation-associated recombination in yeast to generate bacmid AcBac-Syn. The bacmid was then transfected to insect cells and the rescued virus showed similar biological characteristics to the wild-type virus in terms of the kinetics of budded virus production, the morphology of occlusion bodies, and the oral infectivity in insect larvae. For demonstration, a red fluorescent protein gene Dsred was transposed into the attTn7 site by conventional Bac-to-Bac method, and the transfection and infection assays showed that AcBac-Syn can be readily used for foreign gene insertion and expression. AcBac-Syn has several advantages over the conventional AcMNPV bacmids, such as it contains an egfp reporter gene which facilitates visualization of virus propagation and titration; its DNA copy numbers could be induced to a higher level in E. coli; and the retaining of the native polyhedrin gene in the genome making it an attractive system for studying the functions of gene related to occlusion body assembly and oral infection.
Asunto(s)
Baculoviridae , Escherichia coli , Animales , Baculoviridae/genética , Virus ADN , Escherichia coli/genética , Genes Reporteros , Spodoptera , Replicación ViralRESUMEN
Baculoviruses have been widely used as a vector for expressing foreign genes. Among numerous baculoviruses, Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is the most frequently used and it encodes 155 open reading frames (ORFs). Here, we systematically investigated the impact of 42 genes of AcMNPV on the production of infectious budded viruses (BVs) by constructing gene-knockout bacmids and subsequently conducting transfection and infection assays. The results showed that among the 39 functionally unverified genes and 3 recently reported genes, 36 are dispensable for infectious BV production, as the one-step growth curves of the gene-knockout viruses were not significantly different from those of the parental virus. Three genes (ac62, ac82 and ac106/107) are essential for infectious BV production, as deletions thereof resulted in complete loss of infectivity while the repaired viruses showed no significant difference in comparison to the parental virus. In addition, three genes (ac13, ac51 and ac120) are important but not essential for infectious BV production, as gene-knockout viruses produced significantly lower BV levels than that of the parental virus or repaired viruses. We then grouped the 155 AcMNPV genes into three categories (Dispensable, Essential, or Important for infectious BV production). Based on our results and previous publications, we constructed a schematic diagram of a potential mini-genome of AcMNPV, which contains only essential and important genes. The results shed light on our understanding of functional genomics of baculoviruses and provide fundamental information for future engineering of baculovirus expression system.