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.

Total virome characterizations of game animals in China reveals a spectrum of emerging viral pathogens

Game animals are wildlife species often traded and consumed as exotic food, and are potential reservoirs for SARS-CoV and SARS-CoV-2. We performed a meta-transcriptomic analysis of 1725 game animals, representing 16 species and five mammalian orders, sampled across China. From this we identified 71 mammalian viruses, with 45 described for the first time. Eighteen viruses were considered as potentially high risk to humans and domestic animals. Civets (Paguma larvata) carried the highest number of potentially high risk viruses. We identified the transmission of Bat coronavirus HKU8 from a bat to a civet, as well as cross-species jumps of coronaviruses from bats to hedgehogs and from birds to porcupines. We similarly identified avian Influenza A virus H9N2 in civets and Asian badgers, with the latter displaying respiratory symptoms, as well as cases of likely human-to-wildlife virus transmission. These data highlight the importance of game animals as potential drivers of disease emergence. HighlightsO_LI1725 game animals from five mammalian orders were surveyed for viruses C_LIO_LI71 mammalian viruses were discovered, 18 with a potential risk to humans C_LIO_LICivets harbored the highest number of potential high risk viruses C_LIO_LIA species jump of an alphacoronavirus from bats to a civet was identified C_LIO_LIH9N2 influenza virus was detected in a civet and an Asian badger C_LIO_LIHumans viruses were also identified in game animals C_LI

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.

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

Site-specific steric control of SARS-CoV-2 spike glycosylation

A central tenet in the design of vaccines is the display of native-like antigens in the elicitation of protective immunity. The abundance of N-linked glycans across the SARS-CoV-2 spike protein is a potential source of heterogeneity between the many different vaccine candidates under investigation. Here, we investigate the glycosylation of recombinant SARS-CoV-2 spike proteins from five different laboratories and compare them against infectious virus S protein. We find patterns which are conserved across all samples and this can be associated with site-specific stalling of glycan maturation which act as a highly sensitive reporter of protein structure. Molecular dynamics (MD) simulations of a fully glycosylated spike support s a model of steric restrictions that shape enzymatic processing of the glycans. These results suggest that recombinant spike-based SARS-CoV-2 immunogen glycosylation reproducibly recapitulates signatures of viral glycosylation.

Cross-reactive serum and memory B cell responses to spike protein in SARS-CoV-2 and endemic coronavirus infection

Pre-existing immune responses to seasonal endemic coronaviruses could have profound consequences for antibody responses to SARS-CoV-2, either induced in natural infection or through vaccination. Such consequences are well established in the influenza and flavivirus fields. A first step to establish whether pre-existing responses can impact SARS-CoV-2 infection is to understand the nature and extent of cross-reactivity in humans to coronaviruses. We compared serum antibody and memory B cell responses to coronavirus spike (S) proteins from pre-pandemic and SARS-CoV-2 convalescent donors using a series of binding and functional assays. We found weak evidence of pre-existing SARS-CoV-2 cross-reactive serum antibodies in pre-pandemic donors. However, we found stronger evidence of pre-existing cross-reactive memory B cells that were activated on SARS-CoV-2 infection. Monoclonal antibodies (mAbs) isolated from the donors showed varying degrees of cross-reactivity with betacoronaviruses, including SARS and endemic coronaviruses. None of the cross-reactive mAbs were neutralizing except for one that targeted the S2 subunit of the S protein. The results suggest that pre-existing immunity to endemic coronaviruses should be considered in evaluating antibody responses to SARS-CoV-2.

Rapid isolation of potent SARS-CoV-2 neutralizing antibodies and protection in a small animal model

The development of countermeasures to prevent and treat COVID-19 is a global health priority. In under 7 weeks, we enrolled a cohort of SARS-CoV-2-recovered participants, developed neutralization assays to interrogate serum and monoclonal antibody responses, adapted our high throughput antibody isolation, production and characterization pipeline to rapidly screen over 1000 antigen-specific antibodies, and established an animal model to test protection. We report multiple highly potent neutralizing antibodies (nAbs) and show that passive transfer of a nAb provides protection against high-dose SARS-CoV-2 challenge in Syrian hamsters. The study suggests a role for nAbs in prophylaxis, and potentially therapy, of COVID-19. The nAbs define protective epitopes to guide vaccine design.

Comparison of SARS-CoV-2 spike protein binding to human, pet, farm animals, and putative intermediate hosts ACE2 and ACE2 receptors

The emergence of a novel coronavirus, SARS-CoV-2, resulted in a pandemic. Here, we used recently released X-ray structures of human ACE2 bound to the receptor-binding domain (RBD) of the spike protein (S) from SARS-CoV-2 to predict its binding to ACE2 proteins from different animals, including pets, farm animals, and putative intermediate hosts of SARS-CoV-2. Comparing the interaction sites of ACE2 proteins known to serve or not serve as receptor allows to define residues important for binding. From the 20 amino acids in ACE2 that contact S up to seven can be replaced and ACE2 can still function as the SARS-CoV-2 receptor. These variable amino acids are clustered at certain positions, mostly at the periphery of the binding site, while changes of the invariable residues prevent S-binding or infection of the respective animal. Some ACE2 proteins even tolerate the loss or the acquisition of N-glycosylation sites located near the S-interface. Of note, pigs and dogs which are not or not effectively infected, respectively, have only a few changes in the binding site have relatively low levels of ACE2 in the respiratory tract. Comparison of the RBD of S of SARS-CoV-2 with viruses from bat and pangolin revealed that the latter contains only one substitution, whereas the bat virus exhibits five. However, ACE2 of pangolin exhibit seven changes relative to human ACE2, a similar number of substitutions is present in ACE2 of bats, raccoon, and civet suggesting that SARS-CoV-2 may not especially adapted to ACE2 of any of its putative intermediate hosts. These analyses provide new insight into the receptor usage and animal source/origin of SARS-COV-2. IMPORTANCESARS-CoV-2 is threatening people worldwide and there are no drugs or vaccines available to mitigate its spread. The origin of the virus is still unclear and whether pets and livestock can be infected and transmit SARS-CoV-2 are important and unknown scientific questions. Effective binding to the host receptor ACE2 is the first prerequisite for infection of cells and determines the host range. Our analysis provides a framework for the prediction of potential hosts of SARS-CoV-2. We found that ACE2 from species known to support SARS-CoV-2 infection tolerate many amino acid changes indicating that the species barrier might be low. However, the lower expression of ACE2 in the upper respiratory tract of some pets and livestock means more research and monitoring should be done to explore the animal source of infection and the risk of potential cross-species transmission. Finally, the analysis also showed that SARS-CoV-2 may not specifically adapted to any of its putative intermediate hosts.

Clinical observation on the therapeutic effect of warm acupuncture on endometrial cavity fluid from in vitro fertilization-embryo transfer / 中国针灸

OBJECTIVE@#To observe the clinical effect of warm acupuncture on endometrial cavity fluid (ECF) from in vitro fertilization-embryo transfer (IVF-ET), and to explore the mechanism of warm acupuncture on ECF.@*METHODS@#Twenty-nine patients who were prepared for IVF-ET from 2016 to 2019 and whose transplantation was cancelled due to ECF found by vaginal B-ultrasound examination were divided into an observation group (14 cases) and a control group (15 cases) according to random number table method. The warm acupuncture was applied at Zhongwan (CV 12), Qihai (CV 6), Guanyuan (CV 4), Zhongji (CV 3), Guilai (ST 29), Zigong (EX-CA 1), Zusanli (ST 36), Sanyinjiao (SP 6) after the end of menstruation in the observation group, the treatment lasted for 60 min, once a day, 5 times as a course, with 2 days interval between the courses and 3 consecutive courses of treatment were given, until the embryo transfer was performed in the IVF assisted pregnancy cycle. After the end of menstruation, fresh leonurus japonicus capsule was given in the control group, 3 times a day, 0.8 g each time, 7 days as a course, and 3 courses of continuous treatment were received, until the embryo transfer was performed in the IVF assisted pregnancy cycle. The changes of ECF before and after treatment, the time required to prepare for embryo transfer during IVF assisted pregnancy cycle, and the clinical outcome of embryo transfer were observed in the two groups.@*RESULTS@#The decrease of ECF in the observation group was more significant than that in the control group (<0.05). The time required for the embryo transfer in the IVF assisted pregnancy cycle in the observation group was shorter than that in the control group (<0.05). The clinical pregnancy rate in the observation group was 42.9% (6/14), which was significantly higher than 26.7% (4/15) in the control group (<0.05).@*CONCLUSION@#Warm acupuncture may improve the clinical pregnancy rate by raising the local temperature of the lower abdomen, accelerating the blood circulation around the uterus and appendages, promoting the absorption of ECF, improving the uterine environment and endometrial receptivity.

Relation of intermediate-conductance Ca(2+)-activated K(+) channels with ability of proliferation, migration, invasion and IgE secretion of multiple myeloma cells / 中国实验血液学杂志

This study was aimed to investigate the effects of the intermediate-conductance Ca(2+)-activated K(+) (IKCa1) channels on the proliferation, migration, invasion ability and monoclonal immunoglobulin (IgE) secretion of multiple myeloma (MM) cells. Trypan blue exclusion was used to evaluate the impact of clotrimazole (CLO, an inhibitor of the KCa1) on the survival ability of MM cell line U266; transwell chamber and matrigel experiments were used to evaluate the impact of CLO on the ability of the migration and invasion of U266 cells; the influence of CLO on IgE secretion in U266 cells was detected by ELISA. The results showed that small dose of CLO ( ≤ 1.0 µmol/L) could not inhibit the viability of U266 cells. The Transwell and Matrigel invading tests displayed that the cell number moving into lower chamber of transwell decreased after U266 cells treated with small dose of CLO ( ≤ 1.0 µmol/L). After treating the cells with 1.00 µmol/L CLO for 24 h and 48 h, the concentration of IgE in cell supernatant was (4.98 ± 0.39) and (4.38 ± 0.32) ng/ml, while those in control group were (15.41 ± 1.88) and (19.73 ± 2.01) ng/ml, respectively, suggesting significant difference between them(P < 0.05). It is concluded that CLO can decrease the ability of migration and monoclonal immunoglobulin secretion of multiple myeloma cells by blocking the IKCa1, thus this study provides a new think for the targeted therapy of MM.