COVID-19 drives 40% spike in number of people needing humanitarian aid, U.N. says


By: Reuters | New York |

December 1, 2020 12:04:31 pm





“The pandemic has wreaked carnage across the most fragile and vulnerable countries,” he added. (File)

The COVID-19 pandemic has fueled a 40% increase in the number of people needing humanitarian assistance around the globe, the United Nations said on Tuesday, as it appealed for roughly $35 billion to help many of those expected to be in need next year.

“If everyone who will need humanitarian aid next year lived in one country, it would be the world’s fifth largest nation,” U.N. aid chief Mark Lowcock said.

“The pandemic has wreaked carnage across the most fragile and vulnerable countries,” he added.

The United Nations has set out 34 humanitarian response plans covering 56 countries for 2021, aiming to help 160 million of what it forecasts to be 235 million most vulnerable people worldwide facing hunger, conflict and the impacts of climate change and the coronavirus pandemic.

“We always aim to reach about two-thirds of those in need because others, for example the Red Cross, will try to meet the remaining gap,” Lowcock said. He said this year donors gave a record $17 billion to fund humanitarian operations and data showed that aid reached 70% of the people targeted.

While Lowcock noted the $35 billion needed for 2021 was a lot of money, he said it was a “very small” amount compared to what rich countries have spent protecting their citizens during the pandemic.

Key among the concerns for Lowcock is averting famines in countries including Yemen, Afghanistan, northeast Nigeria, South Sudan, Democratic Republic of the Congo and Burkina Faso.

“There is a clear and present danger of really a large scale famine in Yemen now and the single biggest reason for that is because some very important countries who provided a lot of assistance for our relief operation in 2018 and 2019 have not done that in 2020 and those are the countries of the Gulf,” he said.

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Structural analysis of full-length SARS-CoV-2 spike protein from an advanced vaccine candidate


Structure of a vaccine candidate

Much effort is being targeted at developing vaccines that will provide protection against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). A trimeric spike protein that decorates the virus is a primary target of the host immune system and the focus of vaccine development. Bangaru et al. present the structure of a leading vaccine candidate: a full-length spike protein with some modifications aimed at enhancing stability that is formulated in polysorbate 80 detergent. The study confirms that the full-length immunogen is in a stable prefusion conformation and provides a basis for understanding immune responses to the vaccine.

Science, this issue p. 1089

Abstract

Vaccine efforts to combat the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is responsible for the current coronavirus disease 2019 (COVID-19) pandemic, are focused on SARS-CoV-2 spike glycoprotein, the primary target for neutralizing antibodies. We performed cryo–election microscopy and site-specific glycan analysis of one of the leading subunit vaccine candidates from Novavax, which is based on a full-length spike protein formulated in polysorbate 80 detergent. Our studies reveal a stable prefusion conformation of the spike immunogen with slight differences in the S1 subunit compared with published spike ectodomain structures. We also observed interactions between the spike trimers, allowing formation of higher-order spike complexes. This study confirms the structural integrity of the full-length spike protein immunogen and provides a basis for interpreting immune responses to this multivalent nanoparticle immunogen.

Severe acute respiratory syndrome coronavirus (SARS-CoV) caused a global outbreak from 2002 to 2003 (1). Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), from the same lineage of the β-CoV genus as SARS-CoV, recently emerged in China and spread rapidly, infecting more than 28 million people worldwide by September 2020 (2). Coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2, was declared a pandemic by the World Health Organization (WHO). In response, several SARS-CoV-2 vaccine candidates are being developed and tested at various stages of clinical trials (35). The SARS-CoV-2 spike (S) trimeric glycoprotein is a focus of vaccine development because it is the primary target of host immune defenses (5, 6).

Like other type 1 fusion proteins, the SARS-CoV-2 S prefusion trimer is metastable and undergoes structural rearrangement from a prefusion to a postfusion conformation upon S-protein receptor binding and cleavage (7, 8). The structure of the stabilized SARS-CoV-2 spike ectodomain has been solved in its prefusion conformation and resembles the SARS-CoV spike (911). Here, we describe the structure of a leading SARS-CoV-2 S vaccine candidate (NVAX-CoV2373) based on a full-length (FL) S, residues 1 to 1273, which includes the transmembrane (TM) and the cytoplasmic tail (CT) (Fig. 1A). The final construct, SARS-CoV-2-3Q-2P, was also modified at the S1/S2 polybasic cleavage site from RRAR to QQAQ to render it protease resistant, along with two proline substitutions at residues K986 and V987 in the S2 fusion machinery core for enhanced stability (Fig. 1A). The FL spikes, expressed and purified from insect cells, were formulated in 0.01% (v/v) polysorbate 80 (PS 80) detergent. To characterize the structural integrity of the 3Q-2P-FL immunogen, we performed negative-stain electron microscopy of the FL spike constituted in PS 80 in the presence of Matrix-M adjuvant, recapitulating the vaccine formulation being tested in humans. Imaging revealed trimeric spike proteins present as free trimers or as multitrimer rosettes, containing as many as 14 trimers with their TM domains enclosed in micellar cores of PS 80 detergent (Fig. 1B). Tight clustering of the spikes in the NVAX-CoV2373 nanoparticle formulation may lead to stronger immune responses over soluble trimers alone, similar to other viral glycoprotein immunogens (hemagglutinin and respiratory syncytial virus F) (12, 13).

Fig. 1 Evaluation of SARS-CoV-2 3Q-2P-FL spike glycoprotein.

(A) Linear diagram of the sequence and structure elements of the FL SARS-CoV-2 spike protein showing the S1 and S2 ectodomain. Structural elements include a cleavable signal sequence (SS, white), NTD (blue), RBD (green), SD1 and SD2 (light blue), protease cleavage site 2′ (S2′, arrow), fusion peptide (FP, red), heptad repeat 1 (HR1, yellow), central helix (CH, brown), heptad repeat 2 (HR2, purple), TM domain (black), and CT (white). The native furin cleavage site was mutated (RRAR→QQAQ) to be protease resistant and stabilized by introducing two proline (2P) substitutions at positions K986P and V987P to produce SARS-CoV-2 3Q-2P-FL spike. A, Ala; D, Asp; E, Glu; K, Lys; L, Leu; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; V, Val. (B) Representative negative-stain EM images and 2D classes of SARS-CoV-2 3Q-2P-FL, formulated in PS 80 detergent in the presence of Matrix-M adjuvant. In the raw micrograph, spike rosettes are circled in yellow and Matrix-M adjuvant cages are circled in white. 2D classes showing individual spikes, higher-order spike nanoparticles, and Matrix-M cages of different sizes. Matrix-M does not appear to interact with the spike nanoparticles.

We next performed single-particle cryo–electron microscopy (cryo-EM) on the spike formulated in PS 80 detergent (Fig. 2A). Initial two-dimensional (2D) classification revealed the presence of two distinct classes: free spike trimers and dimers of trimers (Fig. 2A). The threefold symmetric (C3) reconstruction of the free spike trimer resulted in a 3.6 Å–resolution map, whereas the asymmetric reconstruction (C1) was refined to 3.8-Å resolution (Fig. 2B and fig. S1, A and B). In previous structures, receptor binding domains (RBDs) exist in either a closed (RBD-down) or an open (RBD-up) conformation that can engage in ACE2 binding (9, 10, 14). By contrast, we observed that all three RBDs on the 3Q-2P-FL spike trimer were in the closed conformation in our reconstructions (Fig. 2B and fig. S1C). Despite the RBD-down conformation, binding analysis of the 3Q-2P-FL immunogen to ACE2 by both biolayer interferometry and enzyme-linked immunosorbent assay clearly shows binding to ACE2, indicating that the RBD is dynamic and the receptor binding site accessible (15). Another study on the prefusion structure of an FL spike protein reported similar findings with RBDs clamped down as a consequence of potential clashes between S2 residues 828 to 853 and subdomain 1 (SD1) when RBD is in open conformation (16). Recent reports by Henderson et al. have revealed that introducing mutations and removing N-linked glycosylation at certain positions can alter the propensity toward “up” and “down” states of the RBD (17, 18).

Fig. 2 Cryo-EM analysis of SARS-CoV-2 3Q-2P-FL spikes.

(A) Representative electron micrograph and 2D class averages of 3Q-2P-FL spikes showing free trimers and complexes of trimers. (B) Side and top views of the B factor–sharpened cryo-EM map of 3Q-2P-FL free trimers showing the spike in prefusion state, with the RBDs in closed conformation. The protomers are colored in blue, green, and coral for clarity. (C) Side and top view of the atomic model of free trimer represented as a ribbon diagram fit into the map density. The protomers are colored in blue, green, and coral, and the map is shown as a transparent gray density. (D) Comparison of 3Q-2P-FL spike with published structures (PDB IDs 6VXX and 6VSB) on a subunit level. PDB 6VXX is shown in cyan, PDB 6VSB in blue, and 3Q-2P-FL spike in coral.

Overall, our cryo-EM map was well resolved in both S1 and S2 subunits (fig. S1D), enabling us to model the full S1 N-terminal domain (NTD) and C-terminal domain (CTD) that were less resolved in previous structures (9, 10). Our final atomic model contains residues 14 to 1146 with breaks only in the flexible loop (619 to 631) and the cleavage site (678 to 688) (Fig. 2C). Superimposition of the coordinate models of 3Q-2P-FL spike with published spike structures [Protein Data Bank (PDB) IDs: 6VXX and 6VSB] revealed substantial domain rearrangements in the S1 subunit of 3Q-2P-FL spike (Fig. 2D). The S1 NTD rotated ~14° relative to published models, whereas the CTD and subdomains showed minor local rearrangements (Fig. 2D). Another recent study also observed differences in NTD conformations at lower pH, although our cryo-EM studies were carried out at neutral pH (19). In our 3Q-2P-FL structure, we observed a shift in residues flanking the 615 to 635 loop, resulting in a salt bridge between residue D614 on one protomer and K854 on a neighboring protomer (Fig. 3A). This observation is particularly notable given the increased prevalence of D614→G (D614G) mutation in the emerging SARS-CoV-2 strains and its potential role in viral transmission and pathogenesis (20). The 615 to 635 loop that is generally disordered in spike trimer structures, including ours, was recently modeled as a helix (PDB ID: 6X6P) (Fig. 3B), although the cryo-EM density (EMD-22078) does not support this assignment (fig. S1E) (11).

Fig. 3 Structural features of the SARS-CoV-2 3Q-2P-FL spike trimer.

(A) Interprotomeric salt-bridge interaction between D614 and K854 in 3Q-2P-FL spike trimer. (B) Comparison of the 615 to 635 loop between 3Q-2P-FL spike shown in coral and PDB 6X6P shown in blue. The residues that were built in 6X6P model but not in our model are shown in dark blue. Threonines at positions 618 and 632 flanking the gap in the 3Q-2P-FL trimer model are shown on both models to highlight their relative positions. T, Thr. (C) Linoleic acid (dark blue) binding within a hydrophobic pocket of one RBD where the fatty acid head group reaches out to interact with the closed RBD of the adjacent protomer. The interacting residues are shown in pink. F, Phe; I, Ile; Y, Tyr. (D) PS 80 detergent (blue) binding within the NTD with potential hydrogen bonding with R190 and H207. The interacting residues are shown in orange. Adjacent protomers are shown in yellow and gray in (A), (C), and (D). H, His.

We observed two additional densities in the S1 subunit that did not correspond to peptide or glycans within the spike (fig. S2A). The first density was buried within a hydrophobic pocket of the CTD (Fig. 3C). We have previously showed palmitoleic acid occupying a similar pocket in the structure of porcine epidemic diarrhea virus (21). This density in SARS-CoV-2 S corresponded to linoleic acid, a polyunsaturated fatty acid; the presence of this ligand was confirmed by mass spectrometry of 3Q-2P-FL spike (fig. S2, B and C). The main chain carboxyl group of linoleic acid interacts with the R408 and Q409 residues of the RBD from the adjacent protomer, potentially stabilizing the observed RBD-down state (Fig. 3C) and consistent with a recent report (22). The second unassigned density, present in the NTD, was larger and more surface exposed than the first (Fig. 3D and fig. S2D). The aliphatic tail of PS 80 fit well into this hydrophobic pocket, whereas the carbonyl and hydroxyl groups were in proximity to residues R190 and H207 with potential for multiple hydrogen bonds between them (Fig. 3D and fig. S2D). The location of the PS 80 ligand provides a possible explanation for the S1 shift seen in our FL trimer density. PS 80 is specific to the formulation of the Novavax 3Q-2P-FL immunogen, but other ligands may also bind this pocket and provide a potential target for drug design against SARS-CoV-2.

Classification of multimeric spike trimer particles yielded two separate classes: a dimer-of-trimers class that reconstructed to a final resolution of 4.5 Å with twofold symmetry and a trimer-of-trimers class that was resolved to 8.0-Å resolution (Fig. 4, A and B, and fig. S3A). In both reconstructions, the interaction between each pair of trimers involved the SD2 of one protomer from each trimer engaging with the NTD of the adjacent trimer (Fig. 4C), with trimer axes tilted 44.5° relative to each other. The dimer-of-trimer interaction was mainly coordinated by the 615 to 635 loop, which, in contrast to the free-trimer structure, was now fully resolved (Fig. 4D). The loop reaches into and induces subtle changes to a pocket on the adjacent NTD compared with the free-trimer model (Fig. 4D). Residues Y145 and H146 in the binding pocket appear to switch positions in the loop-bound state, resulting in a salt-bridge interaction between H146 and D627 and potential stacking between W152 and H146 (Fig. 4E). We also observed minor displacement of residues 68 to 75 and 248 to 250 surrounding the pocket. In the dimer-of-trimers, we also observed N282 glycans at the dimer interface (fig. S3B). As a control, we also performed cryo-EM studies of the SARS-CoV-2-3Q-FL (without 2P). Notably, the structures of the trimers were identical, and we also observed dimers of trimers (fig. S3, C to E)

Fig. 4 Trimer-trimer interactions and glycan analysis.

(A) Side and top views of the sharpened cryo-EM map of 3Q-2P-FL dimers of spike trimers. Individual spike trimers are shown in blue and coral along a twofold axis of symmetry (dotted line). (B) Top view of the B factor–sharpened cryo-EM map of trimer-of-trimers complex with individual trimers colored in blue, coral, and green. (C) Ribbon representation of a protomer from one trimer (blue) interacting with the protomer from the adjacent trimer (coral) docked into the dimers-of-trimers density. (D) A close-up view of the interaction between the protomers of adjacent trimers. One protomer is shown as a ribbon diagram in blue, and its binding partner is shown as surface in gray. Residues 621-PVAIHADQ-628 in the loop with potential interactions to the neighboring NTD are colored yellow, and the residues in the NTD binding pocket are highlighted in coral. Residue D614 at the start of the loop is highlighted in dark blue. Glycosylation at residue 616 is not shown for clarity. G, Gly. (E) Changes occurring in the binding pocket in the bound state (gray) versus the free trimer (pink). Y145 and H146 switch positions to accommodate the loop better, also resulting in salt-bridge formation between H146 and D627. It also results in stacking between W152 and H146. W, Trp. (F) Pseudoviruses expressing SARS-CoV-2 WT or mutant spikes were used to infect HeLa or HeLa-ACE2 cells for 42 to 48 hours. Infection was measured by luciferase intensity RLU (relative light unit) in the lysed cells after infection. (G) Correlation between pseudovirus infection (RLU) and surface expression of SARS-CoV-2 spike variants in 293T cells measured by MFI (mean fluorescence intensity). (H) Site-specific glycan analysis of 3Q-2P-FL spike protein expressed in Sf9 insect cell line. Proportions shown for no occupancy, oligomannose, and complex or paucimannose potential N-linked glycosylation sites (PNGS) are the average and SEM of 3 to 32 distinctive peptides for each glycosite except for sites 17, 709, and 717, where only a single peptide was observed.

Sequence alignment of residues in the 615 to 635 loop and corresponding NTD binding pocket across representative CoV strains belonging to lineage B of betacoronaviruses revealed residues 621-PVAIHADQ-628 are well conserved, but there are notable differences in the binding pocket residues (fig. S4A). Substantial gaps in the interacting NTD loops along with the absence of H146 at the corresponding site on SARS-CoV make it unlikely that SARS-CoV participates in similar intertrimeric interactions. Although the residues in the NTD pocket were almost identical between SARS-CoV-2 and its closely related bat strain Bat-SL-RatG13, we observed some residue differences and one to three amino acid deletions in the loops comprising the NTD binding pocket of representative strains Bat-SL-CoVZC45, BetaCoV/pangolin/Guangdong/1/2019, and BetaCoV/pangolin/Guangxi/P4L/2007 (fig. S4A).

Some human CoVs, including OC43, exclusively use NTD–sialic acid (SA) interactions as their receptor engagement, whereas others such as Middle East respiratory syndrome (MERS) CoV that use the CTD-RBD for primary receptor binding have also been reported to bind SA receptors through their NTD to aid initial attachment to the host cells (2325). Structural comparisons of the SARS-CoV-2 NTD dimerization pocket with that of the SA binding site on MERS spike revealed that they did not coincide with each other (PDB ID: 6Q04) (25) (fig. S4B). Computational and structural studies have proposed residues on SARS-CoV-2 spike that may be involved in SA binding (26, 27). Structural comparison of this putative glycan binding site to the dimerization site revealed them situated adjacent to one another with residues in loop 70 contributing to both the binding pockets (fig. S4C).

We next performed cell surface expression and pseudovirus replication assays with SARS-CoV-2 wild-type (WT) spike and spikes containing mutations in the 615 to 635 loop and NTD pocket. Each residue in the loop 621-PVAIHADQ-628 and residue H146 in the binding pocket were individually mutated to either alanine or glycine. Additionally, we made a spike construct with all eight residues 621-PVAIHADQ-628 replaced with a glycine-serine (GS) linker to completely abrogate binding. Compared with the WT, the mutants generally exhibited lower levels of infectivity (Fig. 4F). Cell surface expression of these mutants in 293T cells revealed that these mutations also disrupted surface expression of the spike protein, with linear correlation between surface expression and pseudovirus replication (Fig. 4G).

Glycans on viral glycoproteins play a wide role in protein folding, stability, and immune recognition and also in facilitating immune evasion. We therefore conducted site-specific glycosylation analysis of the SARS-CoV-2 prefusion spike protein produced in Sf9 insect cells as previously described (28) to assess the extent of glycosylation and the degree of glycan processing from high-mannose or hybrid type to complex type. The analysis detected glycosylation at all 22 N-linked glycan sequons present on SARS-CoV-2 spike (Fig. 4H). Overall, there was high glycan occupancy of >98%, with only two sites (603 and 657) >5% unoccupied. We did not see clear glycan density at either 603 or 657 in the cryo-EM reconstruction of the 3Q-2P-FL spike. Most sites showed extensive glycan processing to complex or paucimannose-type glycans, with only four sites exhibiting ≥40% oligomannose. The glycan analysis also confirmed the presence of glycans at sites 1158, 1173, and 1194 present in the membrane-proximal region of the spike not resolved by cryo-EM. By comparison with site-specific glycan processing of the spike protein produced in mammalian human embryonic kidney (HEK) 293F cells, both mammalian cells and insect cells exhibit extensive processing at most sites. In general, however processing of glycans on the 2019 CoV prefusion spike protein from insect cells was somewhat greater, particularly at sites 709 and 717, which were predominately oligomannose in spike from HEK293 cells but exclusively complex or paucimannose in spike from Sf9 cells (29).

Our structural work is consistent with the burgeoning body of spike structures, albeit with notable differences in the rearrangement of S1 domains and formation of intertrimer interactions (9, 10). Both these findings were seen in the FL spike immunogens assembled into compact and dense nanoparticles. Cryo–electron tomographic reconstructions of intact SARS-CoV-2 virions showed a relatively dispersed distribution of spike protein trimers on the viral surface and no evidence of higher-order aggregates (30). However, another study showed that the D614G mutation present in close proximity to the dimerization loop results in a several-fold increase of spike numbers on the viral surface, resulting in higher spike protein density and a more infectious virion (20). The greater density may be aided by the ability to form such higher-order multimers. Alternatively, the loop that mediates interspike interactions may play a role in viral viability, consistent with our loop mutant data.

Analysis of safety and immunogenicity of the Novavax SARS-CoV-2-3Q-2P-FL immunogen in mice and baboons revealed strong B and T cell responses to the vaccine with no evidence of vaccine-associated enhanced respiratory disease (15). Phase 1 and 2 clinical trial results showed that the vaccine induced immune responses exceeding levels seen in COVID-19 patients (31). Overall, we found that NVAX-CoV2372 is stable, homogeneous, and locked in the antigenically preferred prefusion conformation. With structural, biophysical, and antigenic characterization now complete, ongoing evaluation in humans will provide the true proof-of-principle for this vaccine concept.

References and Notes

  1. N. G. Herrera, N. C. Morano, A. Celikgil, G. I. Georgiev, R. J. Malonis, J. H. Lee, K. Tong, O. Vergnolle, A. B. Massimi, L. Y. Yen, A. J. Noble, M. Kopylov, J. B. Bonanno, S. C. Garrett-Thomson, D. B. Hayes, R. H. Bortz, A. S. Wirchnianski, C. Florez, E. Laudermilch, D. Haslwanter, J. M. Fels, M. E. Dieterle, R. K. Jangra, J. Barnhill, A. Mengotto, D. Kimmel, J. P. Daily, L. A. Pirofski, K. Chandran, M. Brenowitz, S. J. Garforth, E. T. Eng, J. R. Lai, S. C. Almo, Characterization of the SARS-CoV-2 S protein: Biophysical, biochemical, structural, and antigenic analysis. bioRxiv 2020.06.14.150607 [Preprint]. 17 June 2020; .doi:10.1101/2020.06.14.150607

  2. R. Henderson, R. J. Edwards, K. Mansouri, K. Janowska, V. Stalls, M. Kopp, B. F. Haynes, P. Acharya, Glycans on the SARS-CoV-2 spike control the receptor binding domain conformation. bioRxiv 2020.06.26.173765 [Preprint]. 26 June 2020; .doi:10.1101/2020.06.26.173765

  3. T. Zhou, Y. Tsybovsky, A. S. Olia, J. Gorman, M. A. Rapp, G. Cerutti, P. S. Katsamba, A. Nazzari, A. Schon, P. D. Wang, J. Bimela, W. Shi, I. T. Teng, B. Zhang, J. C. Boyington, G. Y. Chuang, J. M. Sampson, M. Sastry, T. Stephens, J. Stuckey, S. Wang, R. A. Friesner, D. D. Ho, J. R. Mascola, L. Shapiro, P. D. Kwong, A pH-dependent switch mediates conformational masking of SARS-CoV-2 spike. bioRxiv 2020.07.04.187989 [Preprint]. 4 July 2020; .doi:10.1101/2020.07.04.187989

  4. L. Zhang, C. B. Jackson, H. Mou, A. Ojha, E. S. Rangarajan, T. Izard, M. Farzan, H. Choe, The D614G mutation in the SARS-CoV-2 spike protein reduces S1 shedding and increases infectivity. bioRxiv 2020.06.12.148726 [Preprint]. 12 June 2020; .doi:10.1101/2020.06.12.148726

  5. R. N. Kirchdoerfer, M. Bhandari, O. Martini, L. M. Sewall, S. Bangaru, K.-J. Yoon, A. B. Ward, Structure and immune recognition of the porcine epidemic diarrhea virus spike protein. bioRxiv 2020.02.18.955195 [Preprint]. 19 February 2020; .doi:10.1101/2020.02.18.955195

Acknowledgments: We thank B. Anderson, H. L. Turner, and C. A. Bowman for their help with electron microscopy, data acquisition, and data processing. We thank B. Webb and L. T. Hoang for their assistance with mass spectrometry and data processing. We thank L. Holden for her assistance with the manuscript. We also thank A. M. Greene at Novavax, Inc., for editing the manuscript. Funding: This work was supported by grants from the National Institute of Allergy and Infectious Diseases Center for HIV/AIDS Vaccine Development (UM1 AI144462 to J.C.P. and A.B.W., R01 AI113867 to J.C.P., R01 AI132317 to D.N., and P01 AI110657 to A.B.W.), the Bill and Melinda Gates Foundation (OPP1170236 to A.B.W.), and Novavax, Inc., Molecular graphics and analyses were performed with UCSF Chimera developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco, with support from National Institutes of Health (R01-GM129325 and P41-GM103311) and the Office of Cyber Infrastructure and Computational Biology, National Institute of Allergy and Infectious Diseases. Author contributions: S.B. and A.B.W. conceived and designed the study. S.B., H.L.T., G.O., and A.A. performed cryo-EM data collection, data processing, and model building. X.W., J.K.D., J.R.Y., and J.C.P. performed site-specific glycan analysis and data interpretation. J.L.T., D.H., and D.N. performed mutagenesis and pseudovirus assays. S.B., G.O., and A.B.W. analyzed and interpreted data. S.B. and A.B.W. wrote the paper, and all authors reviewed and edited the paper. J.H.T., A.D.P., N.P., M.J.M., G.G., and G.S. contributed NVX-CoV2373 and Matrix-M adjuvant and provided advice for sample handling. J.H.T., A.D.P., N.P., M.J.M., G.G., and G.S. also contributed to drafting of the manuscript. Competing interests: Authors J.H.T., A.D.P., N.P., M.J.M., G.G., and G.S. are current employees of Novavax, Inc., a for-profit organization, and these authors own stock or hold stock options. These interests do not alter the authors’ adherence to policies on sharing data and materials. Authors H.L.T. and A.B.W. are inventors on U.S. patent application no. 62/412,703 (“Prefusion Coronavirus Spike Proteins and Their Use”). All other authors have no competing interests to declare. Data and materials availability: The EM maps have been deposited at the Electron Microscopy Data Bank (EMDB) with accession codes EMD-22352 (SARS-CoV-2 3Q-2P-FL spike trimer with C3 symmetry), EMD-22353 (SARS-CoV-2 3Q-2P-FL spike trimer with C1 symmetry), EMD-22354 (SARS-CoV-2 3Q-2P-FL spike dimer-of-trimers with C2 symmetry), EMD-22355 (SARS-CoV-2 3Q-2P-FL spike trimer-of-trimers with C1 symmetry), and EMD-22356 (SARS-CoV-2 3Q-FL spike trimer with C3 symmetry). The atomic models have been deposited at the Protein Data Bank with PDB IDs 7JJI (SARS-CoV-2 3Q-2P-FL spike trimer with C3 symmetry) and 7JJJ (SARS-CoV-2 3Q-2P-FL spike dimer-of-trimers with C2 symmetry). The vaccine construct was provided to the Scripps Research Institute under a Material Transfer Agreement with Novavax, Requests for this material should be addressed to Gale Smith at Novavax. Other materials are available from A.B.W. under a Material Transfer Agreement with the Scripps Research Institute. This work is licensed under a Creative Commons Attribution 4.0 International (CC BY 4.0) license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. To view a copy of this license, visit https://creativecommons.org/licenses/by/4.0/. This license does not apply to figures, photos, artwork, or other content included in the article that is credited to a third party; obtain authorization from the rights holder before using such material.



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Moscow Reports 300% Spike in Coronavirus Deaths


Moscow reported a 300% month-on-month increase in coronavirus deaths for October, the Russian capital’s health department said late Thursday.

In September, the city’s health department said Covid-19 was the direct cause of death among 543 Muscovites.

As the second wave of the virus escalated in October, Moscow’s health department said coronavirus was the main cause in 2,235 deaths that month. That’s 311%, or four times, more than the number in September.

Another 1,338 Muscovites tested positive for Covid-19 but died from other causes in October, the health department said, bringing Moscow’s overall number of coronavirus-related deaths for the month to 3,573. 

A total of 13,718 Muscovites died in October — 2,541 more than in September, 3,262 more than in October 2019 and 3,173 more than the past three-year average. The Moscow health department said the Covid-19 deaths “explain all excess mortality for October.”

Moscow’s overall Covid-19 fatality rate totaled 2.13% when it was the sole cause of death and 4.3% when the disease was not the main cause of death. 

Russia’s coronavirus information center places Moscow’s overall Covid-19 death toll at 8,603 and the national death toll at 38,062, a number widely thought to be undercounted.

The epicenter of Russia’s outbreak, Moscow has registered more than a quarter of the country’s nearly 2.2 million coronavirus cases. Moscow has reported record numbers of Covid-19 infections and hospitalizations in recent days.

Mayor Sergei Sobyanin, who extended some of the remote work and self-isolation restrictions into the new year, said earlier Thursday that the city does not plan to go into full lockdown.



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NT Police confirm property crime spike in Darwin’s rural area, urge residents to share information


Cheryl Vries used to feel so safe in her rural Darwin home, she’d leave the keys in her car’s ignition. Now, she locks every door.

She was one of roughly 300 residents who attended a meeting in Bees Creek on Saturday morning, following a spate of petty crime in the area.

Northern Territory Police confirmed there had been a spike in property crime in the rural area since mid-September, with 34 incidents reported in the past 12 weeks.

But many more questions were raised about why police had not made an arrest.

Ms Vries said she had lived in the rural area for 38 years and frustrations were boiling over.

About 300 people attended the meeting in Bees Creek on Saturday morning, questioning what was being done to curb the spike in property-related crime.(ABC News: Sowaibah Hanife)

‘I would just like to feel safe’

Jenny, who didn’t want to provide her last name, was at the meeting and said while she hadn’t experienced a break in, the stories she’d heard were traumatising.

“I would just like to feel safe in my own home, be able to stay in, walk outside without a problem after 8 o’clock at night,” she said.

“I haven’t slept. I just want something done.”

An angry woman with blonde hair is speaking. She is in a crowd and there are people around her who also look serious.
Residents in Darwin’s rural area said they felt scared in their own homes.(ABC News: Sowaibah Hanifie)

NT Police acting superintendent Paul Faustman said officers were confident their investigation would lead to arrests and believed a group of at least seven people were connected to the crime spree.

“Our indications at the point in time are that these people are targeting … unsecured premises,” he said.

“It appears they’re targeting money, alcohol and cigarettes.”

However, Superintendent Faustman said there was currently insufficient evidence to act and they needed the assistance of the public.

“There appears to be a lot of information out there and they are not sharing that with the Northern Territory Police … by not sharing it, they may be hampering our investigation,” he said.

Paul Faustman is wearing a navy NT Police uniform and talking into a microphone at a meeting. He looks serious.
NT Police Acting Superintendent Paul Faustman said a group of about seven people was believed to be linked to the incidents.(ABC News: Sowaibah Hanifie)

Rural residents want action

NT Police confirmed it had one patrol car dedicated to the Humpty Doo Police Station, but that could be called to Palmerston, about 18 kilometres away, if there was an increase in demand.

“Do you feel supported by our government? Because I don’t feel supported today,” one resident asked police.

Superintendent Faustman said police had made efforts over the past week to ensure the one car allocated to the Humpty Doo station was not diverted elsewhere.

No government minister invited to attend the meeting was present.

Jeanette Kerr is sitting on a chair and listening to someone speak. She has a serious expression and is wearing glasses.
Territory Families deputy CEO Jeanette Kerr told residents children in care were not involved in the crime spree.(ABC News: Sowaibah Hanifie)

Children in care not involved

Many residents expressed concern kids from a youth group home could be involved in the crime spree, but Territory Families said none of the children under the department’s care were suspects.

Deputy chief executive Jeanette Kerr said it was disappointing people often linked crime with children in care.

“While there is a very small percentage of children who have been involved in the youth justice system, that’s not the case here in the rural area,” Ms Kerr said.

“We have one house which is a residential house for three young people who’ve suffered significant trauma, and those young people aren’t involved in the youth justice system.”



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‘Third wave in full swing’: South Korea to close bars, limit restaurants and churches amid new coronavirus spike


South Korea’s capital city and nearby areas will close bars and nightclubs, limit religious gatherings, and restrict service at restaurants, in a bid to contain a burgeoning third wave of coronavirus infections, the health minister said on Sunday.

The Korea Disease Control and Prevention Agency (KDCA) reported 330 new daily coronavirus cases as of midnight on Saturday, a drop from 386 reported the day before, but the fifth straight day of more than 300 new cases.

“The third wave of COVID-19 outbreaks is increasingly in full swing,” Health Minister Park Neung-hoo told a briefing. “The situation is extremely serious and grave.”

A nationwide outbreak was being driven by clusters of infections in the densely-populated Seoul metropolitan area, he said, home to around half of the country’s 52 million residents.

As of Tuesday, major coffee shops in the Seoul area will be required to only offer takeaway and delivery service, while restaurants must close to in-person dining after 9 pm.

Other restrictions will be placed on facilities like gyms, with attendance caps on religious gatherings and sporting events.

Earlier in the day, Prime Minister Chung Sye-kyun told a government meeting that preemptive distancing regulations might be needed to head off a wider outbreak, Yonhap news agency reported.

“We’re at a critical juncture of facing a large number of infections nationwide,” Chung said.

On Saturday, a KDCA official said the country could be facing an outbreak that surpasses two earlier waves of infections, if it fails to block the current spread.

The tightened prevention guidelines are aimed partly at allowing students to go ahead with highly competitive annual college entrance exams scheduled for 3 December.

South Korea has employed an aggressive tracing, testing, and quarantine effort to stamp down outbreaks without imposing lockdowns. But the country has been dogged by a persistent number of small infections, bringing the total number of cases to 30,733 with 505 deaths.

Commuters wearing face masks walk at Shinagawa Station in Tokyo on 19 November, 2020.

Getty Images AsiaPac

Meanwhile in Japan, new COVID-19 cases across the country climbed to a record 2,596 on Saturday, according to public broadcaster NHK.

In Tokyo, the daily infection rate was an all-time high of 539 cases.

The country may reimpose attendance limits for sports and other large events to curb a spike in infections, economy minister Yasutoshi Nishimura said on Sunday.

 

The limits would be applied in areas of the country seeing a sharp increase in cases, Mr Nishimura said on a talk show on NHK.

The government imposed attendance limits earlier in the year but relaxed them in recent months.

People in Australia must stay at least 1.5 metres away from others. Check your jurisdiction’s restrictions on gathering limits.

If you are experiencing cold or flu symptoms, stay home and arrange a test by calling your doctor or contact the Coronavirus Health Information Hotline on 1800 020 080. News and information is available in 63 languages at https://sbs.com.au/coronavirus

Please check the relevant guidelines for your state or territory: NSW,VictoriaQueenslandWestern AustraliaSouth AustraliaNorthern TerritoryACTTasmania



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How to Safely Restart Elective Surgeries After a Covid Spike


The pandemic has wreaked havoc with health care systems’ surgical staffs. Some clinicians are fatigued and stressed, and many are out of practice. They are having to contend with large backlogs of procedures that were postponed. And they have to contend with the ongoing pandemic, with all its uncertainties. To keep surgical staffs and patients safe during these difficult times, health systems should take four steps: make risk visible; ensure that staffs are abiding by existing procedures and protocols and adopting new ones when the need arises; double down on efforts to address psychological safety and the added stress; and be transparent and account for the current environment.

In our work across the country, we are witnessing health care systems in different stages of the Covid-19 pandemic that are trying to address the backlog of elective or nonurgent surgical procedures that were postponed during the initial wave of the pandemic. To safely address it and prepare for future needs to adapt care and priorities based on the ongoing pandemic, systems need to recognize that basic human factors, exacerbated by Covid-19, can threaten the safety of patients and staff and then develop strategies to mitigate them. They include the following:

  • Fatigue. During the pandemic, health care systems redeployed many providers for months to treat Covid-19 patients, and they witnessed extraordinary levels of morbidity and mortality. These providers may be physically and emotionally exhausted.
  • Lack of routine practice. Most surgeons have not practiced at their normal rates (or at all) for several months. As a result, many haven’t had the normal daily or weekly engagement with their technique or their team and need practice to keep up their technical skills; this is most important for surgeons who perform complex procedures. Teams also need to re-establish effective communication patterns.
  • Distraction. Health care systems are creating new procedures and policies because of Covid-19, and staff have not had time to get accustomed and incorporate them into their practices.
  • Overload. Ramping up operating room capacity (some systems are aiming to reach 150%) means surgeries will occur off-peak and at unusual hours. It may also mean that staff are being asked to participate in new or multiple surgical teams. These conditions can make staff more vulnerable to making mistakes or forgetting to take critical steps.
  • Stress. Some providers suffered emotional stress from caring for a deluge of Covid-19 patients, falling ill themselves, or suffering the illness or death of family or friends.

Fortunately, health care systems can identify these threats and mitigate them in order to ensure that surgical teams tackle the backlog of procedures in a failure-free fashion. The following steps, based on reliability science, provide a foundation for doing so:

1. Make risks visible. One of the greatest challenges in safety is not being able to physically see the possible risk prior to a problem arising. The individual surgical team needs to know how it is performing in the moment. In addition to the ongoing monitoring of the patient during the procedure, the surgical team should ask patients questions prior to the procedure to address their physical and mental health and plan for the pre- and post-op care. They cannot assume a patient’s circumstances from six months ago are the same now; patients may be hesitant to disclose changes to their ability to care for themselves or obtain the help they need after surgery.

Insight Center

National medical specialty societies can help make these additional risks visible by redoubling their efforts to get health care systems and physicians to track the high-level outcome measures specific to individual specialties daily, weekly, and monthly. Comparing their current performance to their past performance and that of similar systems in their area will allow them to identify whether safety-related risks are increasing.

Health care system executives should play a significant role in identifying risks and protecting both their workforces and their patients. They must create a dashboard that tracks safety and quality priorities, including adverse events and mortality; make sure it is updated daily; and look at it every day as an executive team. Executives should be able to identify trends in outcomes (positive or negative), be aware of key trigger points, and change course or stop the system if outcomes deteriorate.

2. Honor existing procedures and protocols and adopt new ones as needed. Surgical safety checklists and similar tools must be used with every surgery. We know that these tools help control for human factors and improve surgical outcomes. With increased time pressures there is considerable temptation to move through safety checks quickly and superficially, avoid them altogether, or have them performed by a single person rather than a team. All health care providers and administrators must resist the temptation to cut corners and skip established safety steps as they tackle the backlog of surgeries.

Due to Covid-19, health systems created new precautions to keep patients and staff safe. Prior to turning the surgical system back on at full or increased capacity, executives and care providers at all levels (e.g., frontline leaders and department heads) should ensure that these processes and procedures remain in place. Leaders and surgical care providers should continue to review these new or adapted processes as more is learned about this disease.

3. Double down on efforts that address psychological safety and the added stress. Health care systems should ensure that all supervisors are trained to accurately identify warning signs of psychological stress and make counseling services available to all of their clinical staffs. Some health systems have gone further and have created peer-to-peer “buddy” systems to provide extra support. Others have created mechanisms for clinicians to express a need for a break or further time for preparation without negative consequences.

Leaders of health care systems should ensure that their staffs have a way to raise a concern about an individual provider or provider team, call out a possible safety failure, and, if necessary, “stop the line.” They should make it clear that they not only want them feel safe to do this but also that it is their job to do so and they will be recognized for their vigilance.

4. Be transparent and account for the current environment. Health care systems should share their safety plans and outcome data publicly. Let your patients and their families know that your organization is safe, that you recognize the current environment, and that you are taking every precaution necessary to ensure that patients and family members remain safe with respect to both Covid-19 and the outcome they are expecting from their surgery.

This requires continuous tracking of the prevalence of Covid-19 in the local community (i.e., the positivity rate for the people who have been tested). Health care systems can use the data the states are tracking at the county or city/town level, the rolling weekly average case rate in the communities that the health system serves, and the inpatient case burden (i.e., the number of people currently hospitalized for Covid-19 as compared to overall capacity) both in their own system and in others in their area. Maintaining clear decision trees for when to shut down elective procedural care again and keeping patients and families informed of this possibility is vital to maintaining public confidence.

As health systems continue to adapt to Covid-19 and address the stress it is imposing, their leaders, along with their workforces, must intensify their focus on safety and quality. It requires a strong understanding of human factors — those always present and those exacerbated or created by the pandemic. And it requires implementing processes and procedures that make systems stronger and safer amid new and rapidly changing circumstances. Reopening safely will require attention to reliability science, ensuring physical and psychological safety of the workforce, and continuous monitoring of the state of the local Covid-19 pandemic. Only then will patients, their families, and providers feel safe to return.



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Dallas Asks State to Help Fight Spike in Crime after Cutting $7M from Cop’s Overtime



Texas Governor Greg Abbott is sending state police resources to the City of Dallas in response to a spike in violent crime. The governor responded to a request for assistance from the Dallas Police Department after city leaders cut the police overtime budget by $7 million.

“The rise in violent crime in the city of Dallas is unacceptable, and the Texas Department of Public Safety will assist the Dallas Police Department in their efforts to protect the community and reduce this surge in crime,” Governor Abbott said in a written statement. “Every Texan deserves to feel safe in their own community, and the State of Texas will continue to provide the city of Dallas with the resources they need to crack down on this heinous activity and protect Dallas residents.”

Governor Abbott responded to a request from the Dallas Police Department to provide direct support in reducing violent crime, the governor’s office reported. The Texas Department of Public Safety is sending multiple resources to help the city that is currently in the midst of a spike in violent crime. Those resources include DPS special agents, state troopers, and intelligence analysts. The additional manpower will be directed at helping support gang and drug enforcement operations.

The DPS will also provide two helicopters and two patrol planes to assist in providing air support. Finally, a team of Texas Rangers is being dispatched to support the city’s homicide division.

The call for help comes after the city cut $7 million from the $24 million overtime budget for the Dallas Police Department in September, the Texas Tribune reported. Instead of keeping commissioned officers on the street to attack violent crime, the city chose to divert funds to hiring civilian workers and improve street lighting.

Dallas Mayor Eric Johnson thanked Governor Abbott for sending in additional law enforcement resources, Fox4 News reported.

“I am grateful for the Governor’s willingness to assist Dallas as we combat the unacceptable increases in violent crime in our city. As I said today, this ongoing situation requires an all-hands-on-deck response, and I will continue to push for strategies and partnerships that will reduce crime in our neighborhoods. The people of Dallas deserve our unwavering commitment to their safety.”

“The violent crime in the city is out of hand. I’m tired of it. I’m sick of it,” Mayor Johnson told reporters Wednesday afternoon. “This is not a joke. This is not a game. This is not about being on television. This is about ending the needless and senseless taking of lives in the city.”

Dallas has experienced more nearly 230 homicides this year — seven in the past 24 hours, local media outlets report. Johnson says the city is set to surpass a 16-year high and has already exceeded the total for all of 2019, NBCDFW 5 reported.

Breitbart Texas reached out to the Texas Department of Public Safety for additional information about the deployment of state resources to Dallas. An immediate response was not available.

Bob Price serves as associate editor and senior news contributor for the Breitbart Texas-Border team. He is an original member of the Breitbart Texas team. Price is a regular panelist on Fox 26 Houston’s What’s Your Point? Sunday-morning talk show. Follow him on Twitter @BobPriceBBTX, Parler @BobPrice, and Facebook.





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Fed’s Powell says virus spike threatens U.S. economy – Long Island Business News


Federal Reserve Chair Jerome Powell said Tuesday that the nationwide surge in confirmed coronavirus could slow the economy in the months ahead by discouraging consumers from spending. 

“We’re seeing states begin to impose some activity restrictions,” Powell said in an online discussion with the Bay Area Council, a San Francisco-based business group. “The concern is that people will lose confidence in efforts to control the pandemic, and … we’re seeing signs of that already.” 

While Powell did not elaborate, the government reported earlier Tuesday that retail sales grew just 0.3% in October, the smallest gain since the pandemic sent sales plunging nearly 15% in April. A measure of consumer confidence has also declined this month. 

And JPMorgan says that consumer spending fell earlier this month compared with mid-October, according to activity on 30 million of its credit and debit cards that it anonymously tracks. 

Powell said the threat also means that Congress and the White House should provide more stimulus spending to support the unemployed, states and cities, and small businesses, and to keep the economy afloat. Powell has repeatedly called for more government spending as have many other Fed officials. 

“There hasn’t been a bigger a need for it in a long, long time here,” he said.

He also warned that even when the economy fully recovers, likely after a vaccine is distributed, some industries will likely remain weaker than they were before the pandemic. That could force many of those still unemployed to find work at a new company or in an entirely different industry. Those transitions might also require government support, he said. 

“We’re not going back to the same economy, we’re going back to a different economy,” he said. “That’s going to mean that those people who worked in the service industry, they may need help and support for a time as they find work in new places.”





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