A pseudovirus system enables deep mutational scanning of the full SARS-CoV-2 spike

A major challenge in understanding SARS-CoV-2 evolution is interpreting the antigenic and functional effects of emerging mutations in the viral spike protein. Here we describe a new deep mutational scanning platform based on non-replicative pseudotyped lentiviruses that directly quantifies how large numbers of spike mutations impact antibody neutralization and pseudovirus infection. We demonstrate this new platform by making libraries of the Omicron BA.1 and Delta spikes. These libraries each contain ~7000 distinct amino-acid mutations in the context of up to ~135,000 unique mutation combinations. We use these libraries to map escape mutations from neutralizing antibodies targeting the receptor binding domain, N-terminal domain, and S2 subunit of spike. Overall, this work establishes a high-throughput and safe approach to measure how ~105 combinations of mutations affect antibody neutralization and spike-mediated infection. Notably, the platform described here can be extended to the entry proteins of many other viruses.

Evolution of antibody immunity following Omicron BA.1 breakthrough infection

Understanding the evolution of antibody immunity following heterologous SAR-CoV-2 breakthrough infection will inform the development of next-generation vaccines. Here, we tracked SARS-CoV-2 receptor binding domain (RBD)-specific antibody responses up to six months following Omicron BA.1 breakthrough infection in mRNA-vaccinated individuals. Cross-reactive serum neutralizing antibody and memory B cell (MBC) responses declined by two- to four-fold through the study period. Breakthrough infection elicited minimal de novo Omicron-specific B cell responses but drove affinity maturation of pre-existing cross-reactive MBCs toward BA.1. Public clones dominated the neutralizing antibody response at both early and late time points, and their escape mutation profiles predicted newly emergent Omicron sublineages. The results demonstrate that heterologous SARS-CoV-2 variant exposure drives the evolution of B cell memory and suggest that convergent neutralizing antibody responses continue to shape viral evolution.

A broad and potent neutralization epitope in SARS-related coronaviruses

Many neutralizing antibodies (nAbs) elicited to ancestral SARS-CoV-2 through natural infection and vaccination generally have reduced effectiveness to SARS-CoV-2 variants. Here we show therapeutic antibody ADG20 is able to neutralize all SARS-CoV-2 variants of concern (VOCs) including Omicron (B.1.1.529) as well as other SARS-related coronaviruses. We delineate the structural basis of this relatively escape-resistant epitope that extends from one end of the receptor binding site (RBS) into the highly conserved CR3022 site. ADG20 can then benefit from high potency through direct competition with ACE2 in the more variable RBS and interaction with the more highly conserved CR3022 site. Importantly, antibodies that are able to target this site generally neutralize all VOCs, albeit with reduced potency against Omicron. Thus, this highly conserved and vulnerable site can be exploited for design of universal vaccines and therapeutic antibodies.

Broadly neutralizing anti-S2 antibodies protect against all three human betacoronaviruses that cause severe disease

Pan-betacoronavirus neutralizing antibodies may hold the key to developing broadly protective vaccines against coronaviruses that cause severe disease, for anticipating novel pandemic-causing viruses, and to respond more effectively to SARS-CoV-2 variants. The emergence of the Omicron variant of SARS-CoV-2 has illustrated the limitations of solely targeting the receptor binding domain (RBD) of the envelope Spike (S)-protein. Here, we isolated a large panel of broadly neutralizing antibodies (bnAbs) from SARS-CoV-2 recovered-vaccinated donors that target a conserved S2 region in the fusion machinery on betacoronavirus spikes. Select bnAbs show broad in vivo protection against all three pathogenic betacoronaviruses, SARS-CoV-1, SARS-CoV-2 and MERS-CoV, that have spilled over into humans in the past 20 years to cause severe disease. The bnAbs provide new opportunities for antibody-based interventions and key insights for developing pan-betacoronavirus vaccines.

Engineering SARS-CoV-2 neutralizing antibodies for increased potency and reduced viral escape

The rapid spread of SARS-CoV-2 variants poses a constant threat of escape from monoclonal antibody and vaccine countermeasures. Mutations in the ACE2 receptor binding site on the surface S protein have been shown to disrupt antibody binding and prevent viral neutralization. Here, we use a directed evolution-based approach to engineer three neutralizing antibodies for enhanced binding to S protein. The engineered antibodies showed increased in vitro functional activity in terms of neutralization potency and/or breadth of neutralization against viral variants. Deep mutational scanning revealed that higher binding affinity reduced the total number of viral escape mutations. Studies in the Syrian hamster model showed two examples where the affinity matured antibody provided superior protection compared to the parental antibody. These data suggest that monoclonal antibodies for anti-viral indications could benefit from in vitro affinity maturation to reduce viral escape pathways and appropriate affinity maturation in vaccine immunization could help resist viral variation.

Targeted isolation of panels of diverse human broadly neutralizing antibodies against SARS-like viruses

The emergence of current SARS-CoV-2 variants of concern (VOCs) and potential future spillovers of SARS-like coronaviruses into humans pose a major threat to human health and the global economy 1-7. Development of broadly effective coronavirus vaccines that can mitigate these threats is needed 8, 9. Notably, several recent studies have revealed that vaccination of recovered COVID-19 donors results in enhanced nAb responses compared to SARS-CoV-2 infection or vaccination alone 10-13. Here, we utilized a targeted donor selection strategy to isolate a large panel of broadly neutralizing antibodies (bnAbs) to sarbecoviruses from two such donors. Many of the bnAbs are remarkably effective in neutralization against sarbecoviruses that use ACE2 for viral entry and a substantial fraction also show notable binding to non-ACE2-using sarbecoviruses. The bnAbs are equally effective against most SARS-CoV-2 VOCs and many neutralize the Omicron variant. Neutralization breadth is achieved by bnAb binding to epitopes on a relatively conserved face of the receptor binding domain (RBD) as opposed to strain-specific nAbs to the receptor binding site that are commonly elicited in SARS-CoV-2 infection and vaccination 14-18. Consistent with targeting of conserved sites, select RBD bnAbs exhibited in vivo protective efficacy against diverse SARS-like coronaviruses in a prophylaxis challenge model. The generation of a large panel of potent bnAbs provides new opportunities and choices for next-generation antibody prophylactic and therapeutic applications and, importantly, provides a molecular basis for effective design of pan-sarbecovirus vaccines.

Structural definition of a pan-sarbecovirus neutralizing epitope on the spike S2 subunit.

Three highly pathogenic betacoronaviruses have crossed the species barrier and established human-to-human transmission causing significant morbidity and mortality in the past 20 years. The most current and widespread of these is SARS-CoV-2. The identification of CoVs with zoonotic potential in animal reservoirs suggests that additional outbreaks are likely to occur. Evidence suggests that neutralizing antibodies are important for protection against infection with CoVs. Monoclonal antibodies targeting conserved neutralizing epitopes on diverse CoVs can form the basis for prophylaxis and therapeutic treatments and enable the design of vaccines aimed at providing pan-coronavirus protection. To this end, we previously identified a neutralizing monoclonal antibody, CV3-25 that binds to the SARS-CoV-2 fusion machinery, neutralizes the SARS-CoV-2 Beta variant comparably to the ancestral Wuhan Hu-1 strain, cross neutralizes SARS-CoV-1 and displays cross reactive binding to recombinant proteins derived from the spike-ectodomains of HCoV-OC43 and HCoV-HKU1. Here, we show that the neutralizing activity of CV3-25 is also maintained against the Alpha, Delta and Gamma variants of concern as well as a SARS-CoV-like bat coronavirus with zoonotic potential by binding to a conserved linear peptide in the stem-helix region on sarbecovirus spikes. A 1.74[A] crystal structure of a CV3-25/peptide complex demonstrates that CV3-25 binds to the base of the stem helix at the HR2 boundary to an epitope that is distinct from other stem-helix directed neutralizing mAbs. Thus, CV3-25 defines a novel site of sarbecovirus vulnerability that will inform pan-CoV vaccine development.

Broadly neutralizing antibodies to SARS-related viruses can be readily induced in rhesus macaques

To prepare for future coronavirus (CoV) pandemics, it is desirable to generate vaccines capable of eliciting neutralizing antibody responses against multiple CoVs. Because of the phylogenetic similarity to humans, rhesus macaques are an animal model of choice for many virus-challenge and vaccine-evaluation studies, including SARS-CoV-2. Here, we show that immunization of macaques with SARS-CoV-2 spike (S) protein generates potent receptor binding domain cross- neutralizing antibody (nAb) responses to both SARS-CoV-2 and SARS-CoV-1, in contrast to human infection or vaccination where responses are typically SARS-CoV-2-specific. Furthermore, the macaque nAbs are equally effective against SARS-CoV-2 variants of concern. Structural studies show that different immunodominant sites are targeted by the two primate species. Human antibodies generally target epitopes strongly overlapping the ACE2 receptor binding site (RBS), whereas the macaque antibodies recognize a relatively conserved region proximal to the RBS that represents another potential pan-SARS-related virus site rarely targeted by human antibodies. B cell repertoire differences between the two primates appear to significantly influence the vaccine response and suggest care in the use of rhesus macaques in evaluation of vaccines to SARS-related viruses intended for human use. ONE SENTENCE SUMMARYBroadly neutralizing antibodies to an unappreciated site of conservation in the RBD in SARS- related viruses can be readily induced in rhesus macaques because of distinct properties of the naive macaque B cell repertoire that suggest prudence in the use of the macaque model in SARS vaccine evaluation and design.

A protective broadly cross-reactive human antibody defines a conserved site of vulnerability on beta-coronavirus spikes

Broadly neutralizing antibodies (bnAbs) to coronaviruses (CoVs) are valuable in their own right as prophylactic and therapeutic reagents to treat diverse CoVs and, importantly, as templates for rational pan-CoV vaccine design. We recently described a bnAb, CC40.8, from a coronavirus disease 2019 (COVID-19)-convalescent donor that exhibits broad reactivity with human beta-coronaviruses ({beta}-CoVs). Here, we showed that CC40.8 targets the conserved S2 stem-helix region of the coronavirus spike fusion machinery. We determined a crystal structure of CC40.8 Fab with a SARS-CoV-2 S2 stem-peptide at 1.6 [A] resolution and found that the peptide adopted a mainly helical structure. Conserved residues in {beta}-CoVs interacted with CC40.8 antibody, thereby providing a molecular basis for its broad reactivity. CC40.8 exhibited in vivo protective efficacy against SARS-CoV-2 challenge in two animal models. In both models, CC40.8-treated animals exhibited less weight loss and reduced lung viral titers compared to controls. Furthermore, we noted CC40.8-like bnAbs are relatively rare in human COVID-19 infection and therefore their elicitation may require rational structure-based vaccine design strategies. Overall, our study describes a target on {beta}-CoV spike proteins for protective antibodies that may facilitate the development of pan-{beta}-CoV vaccines. SUMMARYA human mAb isolated from a COVID-19 donor defines a protective cross-neutralizing epitope for pan-{beta}-CoV vaccine design strategies

The application of ultrasound-guided balloon dilatation in treating post-stroke cyclopthyroid achalasia / 中华物理医学与康复杂志

Objective:To observe the effects of applying ultrasound-guided balloon dilatation in the treatment of post-stroke cyclopthyroid achalasia.Methods:Thirty-eight stroke survivors with cyclopneaful achalasia were divided into a control group ( n=19) and an experimental group ( n=19). All had been diagnosed with cycloparyngeal achalasia using videofluoroscopic swallowing study (VFSS). The controls were treated with conventional oral balloon dilatation, while the experimental group underwent ultrasound-guided oral balloon dilatation. The treatment was performed once every 2 days. During the treatment period, the durations and the number of expansions were recorded, and the patient′s comfort and throat pain were evaluated. The Kubota drinking water test (KDWT), standardized swallowing assessment (SSA) scoring and VFSS scoring were compared before and after the 2 weeks of treatment. Results:The average daily duration of expansions in the experimental group was significantly less than in the control group and their number was significantly greater. Comfort and larynx pain were also significantly better in the experimental group. The effective rate in the KDWT was 78.95% in the treatment group, significantly higher than the control group′s 31.58%. Significant differences were also found in the average SSA scores, VFSS pharyngeal phase scores and in the incidence of achalasia between the experimental and control groups.Conclusions:Using ultrasound to guide balloon dilatation can shorten the operation, reduce the throat pain involved and allow for more expansions, greater comfort and thus more effective treatment.

Effects of Siweiyuganzi prescription on anti-peroxidation and blood lipid levels in rats with hyperlipidemia / 中国中西医结合急救杂志

Objective To observe the effects of Siweiyuganzi prescription on anti-peroxidation and blood lipid levels in experimental rats with hyperlipidemia. Methods Sixty male Sprague-Dawley (SD) rats were divided into normal control group, hyperlipidemia model group, Xuezhikang group, Siweiyuganzi prescription large, medium and small dose group according to the random number table method, with 10 rats in each group. The hyperlipidemia rat model was established by intragastric feeding with high fat emulsion everyday 10 mL·kg-1·d-1; normal saline 10 mL/kg was given to the normal control group, twice a day by intragastric feeding; 3 dosages of Siweiyuganzi suspended fluid 12.8, 6.4, 4.3 g·kg-1·d-1 intragastric administrations were given to Siweiyuganzi prescription large, medium and small dose groups respectively; Xuezhikang suspended fluid 0.3 g·kg-1·d-1 was given to Xuezhikang group intragastrically;the same volume of normal saline was given to hyperlipidemia model group. After 4 weeks, the level changes of blood lipid, serum superoxide dismutase (SOD), malonaldehyde (MDA), hydroxymethylglutaryl Coenzyme A (HMG-CoA) were observed. Results Compared to those in the normal control group, the levels of triglyceride (TG), total cholesterol (TC), low density lipoprotein cholesterol (LDL-C), MDA, content and positive expression of HMG-CoA, alanine aminotransferase (ALT) were all higher in hyperlipidemia model group [TG (mmol/L): 6.59±0.72 vs. 4.32±0.36, TC (mmol/L): 7.10±0.25 vs. 5.98±0.40, LDL-C (mmol/L): 4.18±1.30 vs. 2.33±0.35, MDA (μmol/L): 26.05± 5.99 vs. 10.08±1.98, HMG-CoA content (ng/L): 54.60±2.90 vs. 48.73±3.09, HMG-CoA positive expression in liver tissue:(57.80±12.30)% vs. (22.00±4.92)%, ALT (U/L): 106.83±15.75 vs. 81.97±13.18]; SOD and high-density lipoprotein cholesterol (HDL-C) in hyperlipidemia model group were significantly decreased [SOD (kU/L): 295.47±37.51 vs. 345.13±19.76, HDL-C (mmol/L): 2.32±0.49 vs. 4.84±0.45, both P < 0.05]. Compared with the hyperlipidemia model group, the TG, TC, LDL-C, MDA, contents and positive expression of HMG-CoA in each group were significantly reduced, and the SOD and HDL-C were obviously increased, and the changes in the Siweiyuganzi high dose group were more significant than those of the Siweiyuganzi middle-and low-dose groups [TG (mmol/L): 4.70±0.46 vs. 5.40±0.31, 5.70±0.41, TC (mmol/L): 5.80±0.23 vs. 6.14±0.20, 6.56±0.32, LDL-C (mmol/L): 2.56±0.45 vs. 2.93±0.33, 3.28±0.32, HDL-C (mmol/L): 4.58±0.28 vs. 3.89±0.30, 3.59±0.08, SOD (kU/L): 381.45±20.68 vs. 360.60±30.16, 325.49±32.13, MDA (μmol/L): 16.98±5.39 vs. 17.89±5.37, 21.03±6.01, HMG-CoA content (ng/L): 50.58±0.77 vs. 52.16±0.66, 52.90±0.91, HMG-CoA positive expression in liver tissue: (27.90±6.03)% vs. (32.20±7.00)%, (43.00±8.39)%, all P < 0.05]. In the normal control group, there were positive cells scattered in the central vein area and loosely distributed around the portal area in the rat liver; in the hyperlipidemia model group, the positive cells were increased in the central vein area and the cells in relatively great number were seen around the portal area. While the positive cells in Xuezhikang group and in the high, medium and low dose Siweiyuganzi groups were decreased. Conclusion Siweiyuganzi prescription can regulate the levels of blood lipids, prevent and treat the lipid peroxidation caused by hyperlipidemia, and inhibit excessive expression of HMG-CoA in experimental rats with hyperlipidemia.