The diversity of the glycan shield of sarbecoviruses closely related to SARS-CoV-2

The animal reservoirs of sarbecoviruses represent a significant risk of emergent pandemics, as evidenced by the impact of SARS-CoV-2. Vaccines remain successful at limiting severe disease and death, however the continued emergence of SARS-CoV-2 variants, together with the potential for further coronavirus zoonosis, motivates the search for pan-coronavirus vaccines that induce broadly neutralizing antibodies. This necessitates a better understanding of the glycan shields of coronaviruses, which can occlude potential antibody epitopes on spike glycoproteins. Here, we compare the structure of several sarbecovirus glycan shields. Many N-linked glycan attachment sites are shared by all sarbecoviruses, and the processing state of certain sites is highly conserved. However, there are significant differences in the processing state at several glycan sites that surround the receptor binding domain. Our studies reveal similarities and differences in the glycosylation of sarbecoviruses and show how subtle changes in the protein sequence can have pronounced impacts on the glycan shield.

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.

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.

Stereoselective Sc(OTf)3 -Catalyzed Aldol Reactions of Disubstituted Silyl Enol Ethers of Aldehydes with Acetals.

Facile and modular access to stereodefined disubstituted aldehyde-derived silyl enol ethers allowed their successful application in a stereoselective aldol reaction affording the products with excellent yields and diastereomeric ratios. The counter-intuitive stereochemical behavior of this Mukaiyama-aldol reaction is accounted for by a non-classical open transition state.

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.

Regio- and Stereoselective Synthesis of Fully Substituted Silyl Enol Ethers of Ketones and Aldehydes in Acyclic Systems.

The regio- and stereoselective preparation of fully substituted and stereodefined silyl enol ethers of ketones and aldehydes through an allyl-Brook rearrangement is reported. This fast and efficient method proceeds from a mixture of E and Z isomers of easily accessible starting materials.

Platelet-rich plasma vs. steroid injections for hamstring injury-is there really a choice?

OBJECTIVE: To assess the effectiveness of pain relief in patients with grade 2 proximal hamstring injury, treated with platelet-rich plasma (PRP) or corticosteroid injection, by using the primary outcome of visual analog scale (VAS) at 1 week and 4 weeks of follow-up. MATERIALS AND METHODS: A single institution retrospective study was performed for image-guided PRP or steroid injections between 12/1/2015 and 10/30/2017 for proximal hamstring injuries. VAS was measured at 1 week and 4 weeks post-injection via telephone interviews and the pain response was recorded into two groups (negative/no change vs. positive). A comparison of pain responses between PRP and steroid was conducted by generalized estimating equation. RESULTS: Among 56 patients, 32 received PRP and 24 received steroid injections with ages from 13 to 75 years old. At 1 week post-injection follow-up, 23 patients (71.9%) from the PRP group and 11 patients (45.8%) from the steroid group showed positive response. After controlling for age, pre-procedure pain level, and gender, the positive response rate in the PRP group was higher than the steroid group (aOR: 4.04, 95% CI: 1.04-15.63, p value = 0.04). At 4 weeks post-injection, 23 patients (71.9%) from the PRP group and 13 patients (54.2%) from the steroid group showed positive response with no statistical significance (aOR: 2.48, 95% CI: 0.63-9.79, p value = 0.19). CONCLUSIONS: The PRP group had shown more favorable response compared to steroid group at 1 week post-injection, which suggests that PRP therapy can be considered as a conservative treatment choice for grade 2 proximal hamstring injuries with better short-term pain relief based on limited pilot data.

Effects of pressure applied during standardized spinal mobilizations on peripheral skin blood flow: A randomised cross-over study.

BACKGROUND: Peripheral skin blood flow (SBF) changes during and after spinal mobilization (SM), evaluated with laser Doppler flowmetry, may document physiological responses associated with SM. OBJECTIVES: To document variations in SBF during and after application of an SM and evaluate influence of pressure on SBF by applying the same standardized SM with 3 different nonnoxious pressures. DESIGN: Cross-over design with 4 interventions on 4 different days: control (no touch) and 3 SMs applied rhythmically at 5%, 40%, or 80% of pain pressure threshold (sham SM, low-pressure SM, or high-pressure SM, respectively). METHOD: Thirty-two individuals participated. The inspiratory gasp (IG) test was our positive control of vasoconstriction through excitation of the skin sympathetic nervous activity (SSNA). Each session comprised 5 phases: (1) baseline at the end of a 20-min acclimatization, (2) IG test, (3) post-IG phase, (4) SM phase or no manual contact for control, and (5) post-SM phase. A Biopac MP36 system collected SBF data, and a Novel Pliance-X system recorded pressure data. RESULTS/FINDINGS: Equal and significant bilateral vasodilation occurred during application of unilateral sham SM, low-pressure SM, and high-pressure SM. Post-SM significant vasodilation persisted after high-pressure SM. CONCLUSIONS: The current study is the first to describe bilateral peripheral SBF changes occurring during and 5 min after application of standardized SMs. Our post-SM vasodilation suggests involvement of mechanisms other than the putative SSNA-excitatory mechanism proposed with skin conductance measurements. Persistence of post-SM vasodilation following only high-pressure SM suggests possible pressure-dependent mechanisms. However, further research is warranted to clarify our findings.

Assessment of skin blood flow following spinal manual therapy: a systematic review.

Skin blood flow (SBF) indexes have been used to describe physiological mechanisms associated with spinal manual therapy (SMT). The aims of the current review were to assess methods for data collection, assess how investigators interpreted SBF changes, and formulate recommendations to advance manual medicine research. A database search was performed in PubMed, Cochrane Library, the Physiotherapy Evidence Database, and the Cumulative Index to Nursing and Allied Health Literature through April 2014. Articles were included if at least 1 outcome measure was changes in 1 SBF index following SMT. The database search yielded 344 records. Two independent authors applied the inclusion criteria. Twenty studies met the inclusion criteria. Selected studies used heterogeneous methods to assess short-term post-SMT changes in SBF, usually vasoconstriction, which was interpreted as a general sympathoexcitatory effect through central mechanisms. However, this conclusion might be challenged by the current understanding of skin sympathetic nervous activity over local endothelial mechanisms that are specifically controlling SBF. Evaluation of SBF measurements in peripheral tissues following SMT may document physiological responses that are beyond peripheral sympathetic function. Based on the current use of SBF indexes in clinical and physiological research, 14 recommendations for advancing manual medicine research using laser Doppler flowmetry are presented.

Laser Doppler flowmetry in manual medicine research.

Laser Doppler flowmetry (LDF) is commonly used in combination with reactivity tests to noninvasively evaluate skin sympathetic nerve activity and skin microvascular function. In manual medicine research, LDF has been used as a marker for global peripheral sympathetic nervous system function, but these results should be considered with caution because skin sympathetic nerve activity physiology is often overlooked. Another limitation of LDF in manual medicine research is the processing of LDF recordings. Two methods have been suggested: the time-domain analysis and the frequency-domain analysis. Standardization is required for data collection and processing in either domain to accurately interpret these changes in skin blood flow that occur after manual procedures. For physiologic studies using LDF, the authors recommend the use of noninvasive reactivity tests (positive controls) to evaluate the different mechanisms involved in overall skin blood flow changes and to compare the magnitude of these changes with those specifically elicited by manual procedures.