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Cell entry by SARS-CoV-2 requires the binding between the receptor-binding domain (RBD) of the viral Spike protein and the cellular angiotensin-converting enzyme 2 (ACE2). As such, RBD has become the major target for vaccine development, while RBD-specific antibodies are pursued as therapeutics. Here, we report the development and characterization of SARS-CoV-2 RBD-specific VHH/nanobody (Nb) from immunized alpacas. Seven RBD-specific Nbs with high stability were identified using phage display. They bind to SARS-CoV-2 RBD with affinity KD ranging from 2.6 to 113 nM, and six of them can block RBD-ACE2 interaction. The fusion of the Nbs with IgG1 Fc resulted in homodimers with greatly improved RBD-binding affinities (KD ranging from 72.7 pM to 4.5 nM) and nanomolar RBD-ACE2 blocking abilities. Furthermore, fusion of two Nbs with non-overlapping epitopes resulted in hetero-bivalent Nbs, namely aRBD-2-5 and aRBD-2-7, with significantly higher RBD binding affinities (KD of 59.2 pM and 0.25 nM) and greatly enhanced SARS-CoV-2 neutralizing potency. The 50% neutralization dose (ND50) of aRBD-2-5 and aRBD-2-7 was 1.22 ng/mL ([~]0.043 nM) and 3.18 ng/mL ([~]0.111 nM), respectively. These high-affinity SARS-CoV-2 blocking Nbs could be further developed into therapeutics as well as diagnosis reagents for COVID-19. ImportanceTo date, SARS-CoV-2 has caused tremendous loss of human life and economic output worldwide. Although a few COVID-19 vaccines have been approved in several countries, the development of effective therapeutics including SARS-CoV-2 targeting antibodies remains critical. Due to their small size (13-15 kDa), highly solubility and stability, Nbs are particularly well suited for pulmonary delivery and more amenable to engineer into multi-valent formats, compared to the conventional antibody. Here, we report a serial of new anti-SARS-CoV-2 Nbs isolated from immunized alpaca and two engineered hetero-bivalent Nbs. These potent neutralizing Nbs showed promise as potential therapeutics against COVID-19.
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Melanoma differentiation-associated gene-5 (MDA5) acts as a cytoplasmic RNA sensor to detect viral dsRNA and mediates type I interferon (IFN) signaling and antiviral innate immune responses to infection by RNA viruses. Upon recognition of viral dsRNA, MDA5 is activated with K63-linked polyubiquitination and then triggers the recruitment of MAVS and activation of TBK1 and IKK, subsequently leading to IRF3 and NF-{kappa}B phosphorylation. Great numbers of symptomatic and severe infections of SARS-CoV-2 are spreading worldwide, and the poor efficacy of treatment with type I interferon and antiviral agents indicates that SARS-CoV-2 escapes from antiviral immune responses via an unknown mechanism. Here, we report that SARS-CoV-2 nonstructural protein 8 (NSP8) acts as an innate immune suppressor and inhibits type I IFN signaling to promote infection of RNA viruses. It downregulates the expression of type I IFNs, IFN-stimulated genes and proinflammatory cytokines by binding to MDA5 and impairing its K63-linked polyubiquitination. Our findings reveal that NSP8 mediates innate immune evasion during SARS-CoV-2 infection and may serve as a potential target for future therapeutics for SARS-CoV-2 infectious diseases. ImportanceThe large-scale spread of COVID-19 is causing mass casualties worldwide, and the failure of antiviral immune treatment suggests immune evasion. It has been reported that several nonstructural proteins of severe coronaviruses suppress antiviral immune responses; however, the immune suppression mechanism of SARS-CoV-2 remains unknown. Here, we revealed that NSP8 protein of SARS-CoV-2 directly blocks the activation of the cytosolic viral dsRNA sensor MDA5 and significantly downregulates antiviral immune responses. Our study contributes to our understanding of the direct immune evasion mechanism of SARS-CoV-2 by showing that NSP8 suppresses the most upstream sensor of innate immune responses involved in the recognition of viral dsRNA.
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Objective To investigate the curative effects of old age severe chronic obstructive pulmonary disease (COPD) patients inhaled Glucocorticoids with the special devices (Turbuhaler).Methods Seventy-six cases of elderly patients with severe acute exacerbation of COPD patients (53 males,23 females) were divided into experimental group(n =38) and control group(n =38),respectively,according to the random number table.In 10 days of therapy,patients of experimental group inhaled budesonide powder of inhalation 0.2 mg twice daily;Control group inhaled oxygen atomization budesonide suspension 1mg twice daily.The changes of panting symptom were observed.Three months followed-up,the test team inhaled budesonide powder of inhalation 1 mg twice daily,control group stopped inhaling drugs,all patients of panting symptom were observed,also were six-minutes walking distance,the frequency of acute exacerbation and adverse reaction.Intent and compliance program data analysis were performed of the experimental data.Results At the end of the stage of therapy,panting symptom of two group patients were all improved (F =8.746,7.952 respectively,P ＜ 0.05) ; Wheezing score between the two groups 1,5,10 days of treatment differences were not statistically significant(P ＞ 0.05).But the incidence rate of adverse reaction in experimental group was significantly lower than that of control group(x2 =11.372,P ＜0.01),and the treatment costs of test team was significantly lower than that of control group(t =4.573,P ＜ 0.01).At the start of the follow-up period (week 0),two groups of acute exacerbation frequency,wheezing,peak expiratory flow and 6 minutes walking distance had no significant difference(P ＞ 0.05).When follow-up period was at tne end of 12 weeks.The test group of patients with acute exacerbation frequency was significantly lower than the control group(aF within grouP =9.343,aF between groups =16.343,the aF interaction =20.017 ; P ＜ 0.05),wheezing in the control group was significantly mitigate (bF within group =8.537,b F between groups =11.453,bF interaction =13.071 ; P ＜ 0.05),and peak expiratory flow rate than the control group improved significantly (cF within group =9.015,cF between groups =12.426,cF interaction =14.852 ;P ＜ 0.05),6 min walking distance increased significantly compared with the control group (d F within group =12.263,d F between groups =21.371,d F interactive =24.651 ; P ＜ 0.05),the differences were statistically significant.Conclusion Elderly patients with moderate to severe COPD inhaled Glucocorticoids,with the special devices(Turbuhaler),not only in acute exacerbation stage the panting symptom was controlled; but also in stable phase the frequency of acute exacerbation was decreased,and the exercise tolerance was improved.Curative effect is obvious withsafety and well-tolerated.
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Objective To explore the curative effect of inhaled tiotropium upon patients of stable chronic obstructive pulmonary disease (COPD) complicated with pulmonary hypertension.Methods A total of 76 patients (Male/Female =51/25) of stable COPD complicated with pulmonary hypertension were recruited from January 2010 to June 2011.According to table of random digit,they were divided into test group (n =38,M/F =26/12) and control group (n =38,M/F =25/13).Patients of control group inhaled formoterol powder 4.5 μg bid,took oral theophylline sustained-release tablets 0.1 g hid.According to the demand of patient's condition,anti-infective agents and sticky phlegm lyticagent were used.Patients of test group inhaled tiotropium dry powder (18 μg,qd) in addition to the above regimen.All changes in six-minute walk test (SMWT),pulmonary arterial systolic pressure and panting symptom score were recorded at the end of 16 weeks and compared with the baseline.The primary endpoint was the changes in exercise capacity as assessed by SMWT and pulmonary arterial systolic pressure.Safety and tolerance were also investigated.Intent-to-treat and per-protocol analyses were performed.Results After a follow-up period of 16 weeks,both pulmonary arterial systolic pressure and panting symptom score decreased and SMWT walking distance increased (45 ± 16) m in the test group.Compared with the control group,the difference had statistical significance (P ＜ 0.05) and obvious adverse reaction did not occur.Conclusions For patients of stable COPD complicated with pulmonary hypertension,inhaled tiotropium dry powder decreases pulmonary pressure to various extents and improves exercise tolerance and panting symptom.And tolerance is satisfactory.
