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1.
Anal Chem ; 94(15): 5776-5784, 2022 04 19.
Article in English | MEDLINE | ID: covidwho-1882709

ABSTRACT

Characterization of protein glycosylation by tandem mass spectrometry remains challenging owing to the vast diversity of oligosaccharides bound to proteins, the variation in monosaccharide linkage patterns, and the lability of the linkage between the glycan and protein. Here, we have adapted an HCD-triggered-ultraviolet photodissociation (UVPD) approach for the simultaneous localization of glycosites and full characterization of both glycan compositions and intersaccharide linkages, the latter provided by extensive cross-ring cleavages enabled by UVPD. The method is applied to study glycan compositions based on analysis of glycopeptides from proteolytic digestion of recombinant human coronaviruse spike proteins from SARS-CoV-2 and HKU1. UVPD reveals unique intersaccharide linkage information and is leveraged to localize N-linked glycoforms with confidence.


Subject(s)
COVID-19 , Viral Proteins , Glycosylation , Humans , Polysaccharides/chemistry , SARS-CoV-2 , Spike Glycoprotein, Coronavirus , Tandem Mass Spectrometry/methods , Ultraviolet Rays
2.
NPJ Vaccines ; 7(1): 55, 2022 May 23.
Article in English | MEDLINE | ID: covidwho-1860374

ABSTRACT

Adjuvants enhance the magnitude and the durability of the immune response to vaccines. However, there is a paucity of comparative studies on the nature of the immune responses stimulated by leading adjuvant candidates. In this study, we compared five clinically relevant adjuvants in mice-alum, AS03 (a squalene-based adjuvant supplemented with α-tocopherol), AS37 (a TLR7 ligand emulsified in alum), CpG1018 (a TLR9 ligand emulsified in alum), O/W 1849101 (a squalene-based adjuvant)-for their capacity to stimulate immune responses when combined with a subunit vaccine under clinical development. We found that all four of the adjuvant candidates surpassed alum with respect to their capacity to induce enhanced and durable antigen-specific antibody responses. The TLR-agonist-based adjuvants CpG1018 (TLR9) and AS37 (TLR7) induced Th1-skewed CD4+ T cell responses, while alum, O/W, and AS03 induced a balanced Th1/Th2 response. Consistent with this, adjuvants induced distinct patterns of early innate responses. Finally, vaccines adjuvanted with AS03, AS37, and CpG1018/alum-induced durable neutralizing-antibody responses and significant protection against the B.1.351 variant 7 months following immunization. These results, together with our recent results from an identical study in non-human primates (NHPs), provide a comparative benchmarking of five clinically relevant vaccine adjuvants for their capacity to stimulate immunity to a subunit vaccine, demonstrating the capacity of adjuvanted SARS-CoV-2 subunit vaccines to provide durable protection against the B.1.351 variant. Furthermore, these results reveal differences between the widely-used C57BL/6 mouse strain and NHP animal models, highlighting the importance of species selection for future vaccine and adjuvant studies.

3.
Curr Opin Immunol ; 77: 102209, 2022 Aug.
Article in English | MEDLINE | ID: covidwho-1857941

ABSTRACT

Viral proteins fold into a variety of structures as they perform their functions. Structure-based vaccine design aims to exploit knowledge of an antigen's architecture to stabilize it in a vulnerable conformation. We summarize the general principles of structure-based vaccine design, with a focus on the major types of sequence modifications: proline, disulfide, cavity-filling, electrostatic and hydrogen-bond substitution, as well as domain deletion. We then review recent applications of these principles to vaccine-design efforts across five viral families: Coronaviridae, Orthomyxoviridae, Paramyxoviridae, Pneumoviridae, and Filoviridae. Outstanding challenges include continued application of proven design principles to pathogens of interest, as well as development of new strategies for those pathogens that resist traditional techniques.


Subject(s)
Vaccine Development , Viral Proteins , Viral Vaccines , Coronaviridae , Filoviridae , Humans , Orthomyxoviridae , Paramyxoviridae , Pneumovirinae , Viral Proteins/immunology , Viral Vaccines/immunology
4.
Curr Opin Struct Biol ; 74: 102385, 2022 06.
Article in English | MEDLINE | ID: covidwho-1783716

ABSTRACT

Antigen design guided by high-resolution viral glycoprotein structures has successfully generated diverse vaccine candidates for COVID-19. Using conjugation systems to combine antigen design with computationally optimized nanoparticles, researchers have been able to display multivalent antigens with beneficial substitutions that elicited robust humoral immunity with enhanced neutralization potency and breadth. Here, we discuss strategies that have been used for structure-based design and nanoparticle display to develop COVID-19 vaccine candidates as well as potential next-generation vaccine candidates to protect against SARS-CoV-2 variants and other coronaviruses that emerge into the human population.


Subject(s)
COVID-19 , SARS-CoV-2 , COVID-19 Vaccines , Humans , Pandemics/prevention & control , Protein Engineering
5.
Nat Biotechnol ; 40(8): 1270-1275, 2022 Aug.
Article in English | MEDLINE | ID: covidwho-1730301

ABSTRACT

Although several monoclonal antibodies (mAbs) targeting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been approved for coronavirus disease 2019 (COVID-19) therapy, development was generally inefficient, with lead generation often requiring the production and testing of numerous antibody candidates. Here, we report that the integration of target-ligand blocking with a previously described B cell receptor-sequencing approach (linking B cell receptor to antigen specificity through sequencing (LIBRA-seq)) enables the rapid and efficient identification of multiple neutralizing mAbs that prevent the binding of SARS-CoV-2 spike (S) protein to angiotensin-converting enzyme 2 (ACE2). The combination of target-ligand blocking and high-throughput antibody sequencing promises to increase the throughput of programs aimed at discovering new neutralizing antibodies.


Subject(s)
COVID-19 , SARS-CoV-2 , Antibodies, Neutralizing/genetics , Antibodies, Neutralizing/therapeutic use , Antibodies, Viral/genetics , Antibodies, Viral/therapeutic use , Humans , Ligands , Peptidyl-Dipeptidase A , Receptors, Antigen, B-Cell/genetics , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus
6.
Nat Struct Mol Biol ; 29(3): 229-238, 2022 03.
Article in English | MEDLINE | ID: covidwho-1721569

ABSTRACT

Current COVID-19 vaccines and many clinical diagnostics are based on the structure and function of the SARS-CoV-2 spike ectodomain. Using hydrogen-deuterium exchange monitored by mass spectrometry, we have uncovered that, in addition to the prefusion structure determined by cryo-electron microscopy, this protein adopts an alternative conformation that interconverts slowly with the canonical prefusion structure. This new conformation-an open trimer-contains easily accessible receptor-binding domains. It exposes the conserved trimer interface buried in the prefusion conformation, thus exposing potential epitopes for pan-coronavirus antibody and ligand recognition. The population of this state and kinetics of interconversion are modulated by temperature, receptor binding, antibody binding, and sequence variants observed in the natural population. Knowledge of the structure and populations of this conformation will help improve existing diagnostics, therapeutics, and vaccines.


Subject(s)
COVID-19 , Spike Glycoprotein, Coronavirus , Antibodies, Neutralizing , COVID-19 Vaccines , Cryoelectron Microscopy , Epitopes , Humans , Protein Conformation , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/chemistry
8.
Non-conventional in English | MEDLINE, Grey literature | ID: grc-750505

ABSTRACT

With the help of novel processing workflows and algorithms, we have obtained a better understanding of the flexibility and conformational dynamics of the SARS-CoV-2 spike in the prefusion state. We have re-analyzed previous cryo-EM data combining 3D clustering approaches with ways to explore a continuous flexibility space based on 3D Principal Component Analysis. These advanced analyses revealed a concerted motion involving the receptor-binding domain (RBD), N-terminal domain (NTD), and subdomain 1 and 2 (SD1 & SD2) around the previously characterized 1-RBD-up state, which have been modeled as elastic deformations. We show that in this dataset there are not well-defined, stable, spike conformations, but virtually a continuum of states moving in a concerted fashion. We obtained an improved resolution ensemble map with minimum bias, from which we model by flexible fitting the extremes of the change along the direction of maximal variance. Moreover, a high-resolution structure of a recently described biochemically stabilized form of the spike is shown to greatly reduce the dynamics observed for the wild-type spike. Our results provide new detailed avenues to potentially restrain the spike dynamics for structure-based drug and vaccine design and at the same time give a warning of the potential image processing classification instability of these complicated datasets, having a direct impact on the interpretability of the results.

9.
J Mol Biol ; 434(2): 167332, 2022 01 30.
Article in English | MEDLINE | ID: covidwho-1492301

ABSTRACT

Extensive glycosylation of viral glycoproteins is a key feature of the antigenic surface of viruses and yet glycan processing can also be influenced by the manner of their recombinant production. The low yields of the soluble form of the trimeric spike (S) glycoprotein from SARS-CoV-2 has prompted advances in protein engineering that have greatly enhanced the stability and yields of the glycoprotein. The latest expression-enhanced version of the spike incorporates six proline substitutions to stabilize the prefusion conformation (termed SARS-CoV-2 S HexaPro). Although the substitutions greatly enhanced expression whilst not compromising protein structure, the influence of these substitutions on glycan processing has not been explored. Here, we show that the site-specific N-linked glycosylation of the expression-enhanced HexaPro resembles that of an earlier version containing two proline substitutions (2P), and that both capture features of native viral glycosylation. However, there are site-specific differences in glycosylation of HexaPro when compared to 2P. Despite these discrepancies, analysis of the serological reactivity of clinical samples from infected individuals confirmed that both HexaPro and 2P protein are equally able to detect IgG, IgA, and IgM responses in all sera analysed. Moreover, we extend this observation to include an analysis of glycan engineered S protein, whereby all N-linked glycans were converted to oligomannose-type and conclude that serological activity is not impacted by large scale changes in glycosylation. These observations suggest that variations in glycan processing will not impact the serological assessments currently being performed across the globe.


Subject(s)
Antibodies, Viral/immunology , COVID-19/immunology , Mutation, Missense/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Antibodies, Viral/blood , Binding Sites/genetics , COVID-19/virology , Glycosylation , Humans , Immunoglobulin A/blood , Immunoglobulin A/immunology , Immunoglobulin G/blood , Immunoglobulin G/immunology , Immunoglobulin M/blood , Immunoglobulin M/immunology , Mannose/metabolism , Mutation, Missense/genetics , Oligosaccharides/metabolism , Polysaccharides/metabolism , Proline/genetics , Proline/immunology , Proline/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/immunology , Recombinant Proteins/metabolism , SARS-CoV-2/metabolism , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism
10.
Sci Immunol ; 6(64): eabj2901, 2021 Oct 15.
Article in English | MEDLINE | ID: covidwho-1470496

ABSTRACT

The introduction of vaccines has inspired hope in the battle against SARS-CoV-2. However, the emergence of viral variants, in the absence of potent antivirals, has left the world struggling with the uncertain nature of this disease. Antibodies currently represent the strongest correlate of immunity against SARS-CoV-2, thus we profiled the earliest humoral signatures in a large cohort of acutely ill (survivors and nonsurvivors) and mild or asymptomatic individuals with COVID-19. Although a SARS-CoV-2­specific immune response evolved rapidly in survivors of COVID-19, nonsurvivors exhibited blunted and delayed humoral immune evolution, particularly with respect to S2-specific antibodies. Given the conservation of S2 across ß-coronaviruses, we found that the early development of SARS-CoV-2­specific immunity occurred in tandem with preexisting common ß-coronavirus OC43 humoral immunity in survivors, which was also selectively expanded in individuals that develop a paucisymptomatic infection. These data point to the importance of cross-coronavirus immunity as a correlate of protection against COVID-19.


Subject(s)
COVID-19/immunology , Cross Reactions , Immunity, Humoral , SARS-CoV-2/immunology , Adolescent , Cohort Studies , Coronavirus OC43, Human/immunology , Disease Progression , Humans , Immunoglobulin Class Switching , Receptors, Fc/immunology , Spike Glycoprotein, Coronavirus/immunology , Survivors , Young Adult
11.
Cell Rep ; 37(5): 109929, 2021 11 02.
Article in English | MEDLINE | ID: covidwho-1466097

ABSTRACT

Current coronavirus (CoV) vaccines primarily target immunodominant epitopes in the S1 subunit, which are poorly conserved and susceptible to escape mutations, thus threatening vaccine efficacy. Here, we use structure-guided protein engineering to remove the S1 subunit from the Middle East respiratory syndrome (MERS)-CoV spike (S) glycoprotein and develop stabilized stem (SS) antigens. Vaccination with MERS SS elicits cross-reactive ß-CoV antibody responses and protects mice against lethal MERS-CoV challenge. High-throughput screening of antibody-secreting cells from MERS SS-immunized mice led to the discovery of a panel of cross-reactive monoclonal antibodies. Among them, antibody IgG22 binds with high affinity to both MERS-CoV and severe acute respiratory syndrome (SARS)-CoV-2 S proteins, and a combination of electron microscopy and crystal structures localizes the epitope to a conserved coiled-coil region in the S2 subunit. Passive transfer of IgG22 protects mice against both MERS-CoV and SARS-CoV-2 challenge. Collectively, these results provide a proof of principle for cross-reactive CoV antibodies and inform the development of pan-CoV vaccines and therapeutic antibodies.


Subject(s)
Antibodies, Viral/immunology , Middle East Respiratory Syndrome Coronavirus/immunology , Spike Glycoprotein, Coronavirus/immunology , Animals , Cell Line , Coronavirus Infections/immunology , Coronavirus Infections/prevention & control , Cross Reactions , Drug Design , Epitope Mapping , Female , Immunoglobulin G/immunology , Male , Mice , Mice, Inbred BALB C , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/chemistry , Viral Vaccines/immunology
12.
Nat Protoc ; 16(11): 5339-5356, 2021 11.
Article in English | MEDLINE | ID: covidwho-1454802

ABSTRACT

The severe acute respiratory syndrome coronavirus 2 spike protein is a critical component of coronavirus disease 2019 vaccines and diagnostics and is also a therapeutic target. However, the spike protein is difficult to produce recombinantly because it is a large trimeric class I fusion membrane protein that is metastable and heavily glycosylated. We recently developed a prefusion-stabilized spike variant, termed HexaPro for six stabilizing proline substitutions, that can be expressed with a yield of >30 mg/L in ExpiCHO cells. This protocol describes an optimized workflow for expressing and biophysically characterizing rationally engineered spike proteins in Freestyle 293 and ExpiCHO cell lines. Although we focus on HexaPro, this protocol has been used to purify over a hundred different spike variants in our laboratories. We also provide guidance on expression quality control, long-term storage, and uses in enzyme-linked immunosorbent assays. The entire protocol, from transfection to biophysical characterization, can be completed in 7 d by researchers with basic tissue cell culture and protein purification expertise.


Subject(s)
Gene Expression Regulation, Viral/physiology , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Animals , CHO Cells , Cricetinae , Cricetulus , HEK293 Cells , Humans , Models, Molecular , Protein Conformation
13.
Cell ; 184(21): 5432-5447.e16, 2021 10 14.
Article in English | MEDLINE | ID: covidwho-1454060

ABSTRACT

Understanding vaccine-elicited protection against SARS-CoV-2 variants and other sarbecoviruses is key for guiding public health policies. We show that a clinical stage multivalent SARS-CoV-2 spike receptor-binding domain nanoparticle (RBD-NP) vaccine protects mice from SARS-CoV-2 challenge after a single immunization, indicating a potential dose-sparing strategy. We benchmarked serum neutralizing activity elicited by RBD-NPs in non-human primates against a lead prefusion-stabilized SARS-CoV-2 spike (HexaPro) using a panel of circulating mutants. Polyclonal antibodies elicited by both vaccines are similarly resilient to many RBD residue substitutions tested, although mutations at and surrounding position 484 have negative consequences for neutralization. Mosaic and cocktail nanoparticle immunogens displaying multiple sarbecovirus RBDs elicit broad neutralizing activity in mice and protect mice against SARS-CoV challenge even in the absence of SARS-CoV RBD in the vaccine. This study provides proof of principle that multivalent sarbecovirus RBD-NPs induce heterotypic protection and motivates advancing such broadly protective sarbecovirus vaccines to the clinic.

14.
mBio ; 12(5): e0247321, 2021 10 26.
Article in English | MEDLINE | ID: covidwho-1450586

ABSTRACT

Most known SARS-CoV-2 neutralizing antibodies (nAbs), including those approved by the FDA for emergency use, inhibit viral infection by targeting the receptor-binding domain (RBD) of the spike (S) protein. Variants of concern (VOC) carrying mutations in the RBD or other regions of S reduce the effectiveness of many nAbs and vaccines by evading neutralization. Therefore, therapies that are less susceptible to resistance are urgently needed. Here, we characterized the memory B-cell repertoire of COVID-19 convalescent donors and analyzed their RBD and non-RBD nAbs. We found that many of the non-RBD-targeting nAbs were specific to the N-terminal domain (NTD). Using neutralization assays with authentic SARS-CoV-2 and a recombinant vesicular stomatitis virus carrying SARS-CoV-2 S protein (rVSV-SARS2), we defined a panel of potent RBD and NTD nAbs. Next, we used a combination of neutralization-escape rVSV-SARS2 mutants and a yeast display library of RBD mutants to map their epitopes. The most potent RBD nAb competed with hACE2 binding and targeted an epitope that includes residue F490. The most potent NTD nAb epitope included Y145, K150, and W152. As seen with some of the natural VOC, the neutralization potencies of COVID-19 convalescent-phase sera were reduced by 4- to 16-fold against rVSV-SARS2 bearing Y145D, K150E, or W152R spike mutations. Moreover, we found that combining RBD and NTD nAbs did not enhance their neutralization potential. Notably, the same combination of RBD and NTD nAbs limited the development of neutralization-escape mutants in vitro, suggesting such a strategy may have higher efficacy and utility for mitigating the emergence of VOC. IMPORTANCE The U.S. FDA has issued emergency use authorizations (EUAs) for multiple investigational monoclonal antibody (MAb) therapies for the treatment of mild to moderate COVID-19. These MAb therapeutics are solely targeting the receptor-binding domain of the SARS-CoV-2 spike protein. However, the N-terminal domain of the spike protein also carries crucial neutralizing epitopes. Here, we show that key mutations in the N-terminal domain can reduce the neutralizing capacity of convalescent-phase COVID-19 sera. We report that a combination of two neutralizing antibodies targeting the receptor-binding and N-terminal domains may be beneficial to combat the emergence of virus variants.


Subject(s)
Antibodies, Neutralizing/immunology , COVID-19/genetics , COVID-19/immunology , Mutation/immunology , RNA-Binding Motifs/immunology , SARS-CoV-2/genetics , SARS-CoV-2/immunology , Humans , Neutralization Tests
15.
Cell Rep ; 37(1): 109784, 2021 10 05.
Article in English | MEDLINE | ID: covidwho-1442299

ABSTRACT

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) lineages that are more transmissible and resistant to currently approved antibody therapies poses a considerable challenge to the clinical treatment of coronavirus disease (COVID-19). Therefore, the need for ongoing discovery efforts to identify broadly reactive monoclonal antibodies to SARS-CoV-2 is of utmost importance. Here, we report a panel of SARS-CoV-2 antibodies isolated using the linking B cell receptor to antigen specificity through sequencing (LIBRA-seq) technology from an individual who recovered from COVID-19. Of these antibodies, 54042-4 shows potent neutralization against authentic SARS-CoV-2 viruses, including variants of concern (VOCs). A cryoelectron microscopy (cryo-EM) structure of 54042-4 in complex with the SARS-CoV-2 spike reveals an epitope composed of residues that are highly conserved in currently circulating SARS-CoV-2 lineages. Further, 54042-4 possesses uncommon genetic and structural characteristics that distinguish it from other potently neutralizing SARS-CoV-2 antibodies. Together, these findings provide motivation for the development of 54042-4 as a lead candidate to counteract current and future SARS-CoV-2 VOCs.


Subject(s)
Angiotensin-Converting Enzyme 2/immunology , Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/immunology , COVID-19/immunology , SARS-CoV-2/chemistry , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Angiotensin-Converting Enzyme 2/chemistry , Animals , Antibodies, Viral/immunology , Antibody Formation , COVID-19/genetics , COVID-19/virology , Cell Line , Chlorocebus aethiops , Cryoelectron Microscopy , Epitope Mapping/methods , Epitopes/chemistry , Epitopes/immunology , High-Throughput Screening Assays/methods , Humans , Male , Middle Aged , Protein Binding , Protein Interaction Domains and Motifs , Receptors, Antigen, B-Cell/chemistry , Receptors, Antigen, B-Cell/immunology , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/chemistry , Vero Cells
16.
Nat Commun ; 12(1): 5652, 2021 09 27.
Article in English | MEDLINE | ID: covidwho-1440473

ABSTRACT

The emergence of numerous variants of SARS-CoV-2, the causative agent of COVID-19, has presented new challenges to the global efforts to control the COVID-19 pandemic. Here, we obtain two cross-neutralizing antibodies (7D6 and 6D6) that target Sarbecoviruses' receptor-binding domain (RBD) with sub-picomolar affinities and potently neutralize authentic SARS-CoV-2. Crystal structures show that both antibodies bind a cryptic site different from that recognized by existing antibodies and highly conserved across Sarbecovirus isolates. Binding of these two antibodies to the RBD clashes with the adjacent N-terminal domain and disrupts the viral spike. Both antibodies confer good resistance to mutations in the currently circulating SARS-CoV-2 variants. Thus, our results have direct relevance to public health as options for passive antibody therapeutics and even active prophylactics. They can also inform the design of pan-sarbecovirus vaccines.


Subject(s)
Antibodies, Viral/immunology , Broadly Neutralizing Antibodies/immunology , COVID-19/therapy , Immunization, Passive/methods , SARS-CoV-2/immunology , Animals , Antibodies, Monoclonal/administration & dosage , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/isolation & purification , Antibodies, Monoclonal/metabolism , Antibodies, Viral/administration & dosage , Antibodies, Viral/isolation & purification , Antibodies, Viral/metabolism , Binding Sites/genetics , Binding Sites/immunology , Broadly Neutralizing Antibodies/administration & dosage , Broadly Neutralizing Antibodies/isolation & purification , Broadly Neutralizing Antibodies/metabolism , CHO Cells , COVID-19/epidemiology , COVID-19/immunology , COVID-19/virology , Chlorocebus aethiops , Cricetulus , Epitopes/immunology , HEK293 Cells , Humans , Mice , Middle East Respiratory Syndrome Coronavirus/genetics , Middle East Respiratory Syndrome Coronavirus/immunology , Neutralization Tests , Pandemics/prevention & control , Protein Multimerization , Receptors, Virus/metabolism , SARS-CoV-2/genetics , Sf9 Cells , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/metabolism , Vero Cells
18.
Science ; 372(6546): 1108-1112, 2021 06 04.
Article in English | MEDLINE | ID: covidwho-1388437

ABSTRACT

The molecular composition and binding epitopes of the immunoglobulin G (IgG) antibodies that circulate in blood plasma after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection are unknown. Proteomic deconvolution of the IgG repertoire to the spike glycoprotein in convalescent subjects revealed that the response is directed predominantly (>80%) against epitopes residing outside the receptor binding domain (RBD). In one subject, just four IgG lineages accounted for 93.5% of the response, including an amino (N)-terminal domain (NTD)-directed antibody that was protective against lethal viral challenge. Genetic, structural, and functional characterization of a multidonor class of "public" antibodies revealed an NTD epitope that is recurrently mutated among emerging SARS-CoV-2 variants of concern. These data show that "public" NTD-directed and other non-RBD plasma antibodies are prevalent and have implications for SARS-CoV-2 protection and antibody escape.


Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19/immunology , Immunoglobulin G/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Animals , Antibodies, Monoclonal/blood , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/blood , Antibodies, Neutralizing/chemistry , Antibodies, Viral/blood , Antibodies, Viral/chemistry , Antibody Affinity , COVID-19/prevention & control , Epitopes/immunology , Humans , Immune Evasion , Immunoglobulin G/blood , Immunoglobulin G/chemistry , Immunoglobulin Heavy Chains/immunology , Immunoglobulin Variable Region/immunology , Mice , Mice, Inbred BALB C , Mutation , Protein Domains , Proteomics , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics
20.
Nat Commun ; 12(1): 1240, 2021 02 23.
Article in English | MEDLINE | ID: covidwho-1387330

ABSTRACT

Cryo-electron microscopy (cryo-EM) maps usually show heterogeneous distributions of B-factors and electron density occupancies and are typically B-factor sharpened to improve their contrast and interpretability at high-resolutions. However, 'over-sharpening' due to the application of a single global B-factor can distort processed maps causing connected densities to appear broken and disconnected. This issue limits the interpretability of cryo-EM maps, i.e. ab initio modelling. In this work, we propose 1) approaches to enhance high-resolution features of cryo-EM maps, while preventing map distortions and 2) methods to obtain local B-factors and electron density occupancy maps. These algorithms have as common link the use of the spiral phase transformation and are called LocSpiral, LocBSharpen, LocBFactor and LocOccupancy. Our results, which include improved maps of recent SARS-CoV-2 structures, show that our methods can improve the interpretability and analysis of obtained reconstructions.

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