Biology subjects

Edwards, R. J.

Publications and source records attributed to Edwards, R. J..

13 recordsLinked to original sources

bioRxiv2021

Effect of natural mutations of SARS-CoV-2 on spike structure, conformation and antigenicity

New SARS-CoV-2 variants that have accumulated multiple mutations in the spike (S) glycoprotein enable increased transmission and resistance to neutralizing antibodies. Here, we study the antigenic and structural impacts of the S protein mutations from four variants, one that was involved in transmission between minks and humans, and three that rapidly spread in human populations and originated in the United Kingdom, Brazil or South Africa. All variants either retained or improved binding to the ACE2 receptor. The B.1.1.7 (UK) and B.1.1.28 (Brazil) spike variants showed reduced binding to neutralizing NTD and RBD antibodies, respectively, while the B.1.351 (SA) variant showed reduced binding to both NTD- and RBD-directed antibodies. Cryo-EM structural analyses revealed allosteric effects of the mutations on spike conformations and revealed mechanistic differences that either drive inter-species transmission or promotes viral escape from dominant neutralizing epitopes. HighlightsO_LICryo-EM structures reveal changes in SARS-CoV-2 S protein during inter-species transmission or immune evasion. C_LIO_LIAdaptation to mink resulted in increased ACE2 binding and spike destabilization. C_LIO_LIB.1.1.7 S mutations reveal an intricate balance of stabilizing and destabilizing effects that impact receptor and antibody binding. C_LIO_LIE484K mutation in B.1.351 and B.1.1.28 S proteins drives immune evasion by altering RBD conformation. C_LIO_LIS protein uses different mechanisms to converge upon similar solutions for altering RBD up/down positioning. C_LI

microbiology↗

bioRxiv2021

SARS-CoV-2 vaccination induces neutralizing antibodies against pandemic and pre-emergent SARS-related coronaviruses in monkeys

Betacoronaviruses (betaCoVs) caused the severe acute respiratory syndrome (SARS) and Middle East Respiratory Syndrome (MERS) outbreaks, and now the SARS-CoV-2 pandemic. Vaccines that elicit protective immune responses against SARS-CoV-2 and betaCoVs circulating in animals have the potential to prevent future betaCoV pandemics. Here, we show that immunization of macaques with a multimeric SARS-CoV-2 receptor binding domain (RBD) nanoparticle adjuvanted with 3M-052-Alum elicited cross-neutralizing antibody responses against SARS-CoV-1, SARS-CoV-2, batCoVs and the UK B.1.1.7 SARS-CoV-2 mutant virus. Nanoparticle vaccination resulted in a SARS-CoV-2 reciprocal geometric mean neutralization titer of 47,216, and robust protection against SARS-CoV-2 in macaque upper and lower respiratory tracts. Importantly, nucleoside-modified mRNA encoding a stabilized transmembrane spike or monomeric RBD protein also induced SARS-CoV-1 and batCoV cross-neutralizing antibodies, albeit at lower titers. These results demonstrate current mRNA vaccines may provide some protection from future zoonotic betaCoV outbreaks, and provide a platform for further development of pan-betaCoV nanoparticle vaccines.

immunology↗

bioRxiv2021

The functions of SARS-CoV-2 neutralizing and infection-enhancing antibodies in vitro and in mice and nonhuman primates

SARS-CoV-2 neutralizing antibodies (NAbs) protect against COVID-19. A concern regarding SARS-CoV-2 antibodies is whether they mediate disease enhancement. Here, we isolated NAbs against the receptor-binding domain (RBD) and the N-terminal domain (NTD) of SARS-CoV-2 spike from individuals with acute or convalescent SARS-CoV-2 or a history of SARS-CoV-1 infection. Cryo-electron microscopy of RBD and NTD antibodies demonstrated function-specific modes of binding. Select RBD NAbs also demonstrated Fc receptor-{gamma} (Fc{gamma}R)-mediated enhancement of virus infection in vitro, while five non-neutralizing NTD antibodies mediated Fc{gamma}R-independent in vitro infection enhancement. However, both types of infection-enhancing antibodies protected from SARS-CoV-2 replication in monkeys and mice. Nonetheless, three of 31 monkeys infused with enhancing antibodies had higher lung inflammation scores compared to controls. One monkey had alveolar edema and elevated bronchoalveolar lavage inflammatory cytokines. Thus, while in vitro antibody-enhanced infection does not necessarily herald enhanced infection in vivo, increased lung inflammation can occur in SARS-CoV-2 antibody-infused macaques.

immunology↗

bioRxiv2020

IgM Natural Antibodies Bind HLA-E-Leader Peptide Complexes and Modulate NK Cell Cytotoxicity

HLA-E is a non-classical class Ib molecule that has limited polymorphism and binds HLA class Ia leader peptides (VL9). HLA-E-VL9 complexes interact with the natural killer (NK) cell receptors NKG2A-C/CD94 and regulate NK cell-mediated cytotoxicity. Here we isolated a murine IgM antibody 3H4, that specifically recognized HLA-E-VL9 bound complexes and enhanced killing of HLA-E-VL9-expressing cells by an NKG2A+ NK cell line. Structural analysis revealed how 3H4 prevents CD94/NKG2A docking on HLA-E-VL9 by binding with an overlapping footprint. Upon in vitro maturation, an affinity-optimized 3H4 IgG showed enhanced NK killing of HLA-E-VL9-expressing cells. Remarkably, HLA-E-VL9-specific IgM autoantibodies with similar specificity and functions to 3H4 were subsequently isolated from naive B cells of cytomegalovirus (CMV)-negative, healthy male human donors. Thus, a repertoire of germline low affinity HLA-E-VL9-reactive antibodies are present in both naive human and murine B cell repertoires. These antibodies can enhance NK cell cytotoxicity and therefore have potential for therapeutic modulation of NK cell function.

immunology↗

bioRxiv2020

Desert Dingo (Canis lupus dingo) genome provides insights into their role in the Australian ecosystem.

The dingo is Australias iconic top-order predator and arrived on the continent between 5,000-8,000 years ago. To provide an unbiased insight into its evolutionary affiliations and biological interactions, we coupled long-read DNA sequencing with a multiplatform scaffolding approach to produce an ab initio genome assembly of the desert dingo (85X coverage) we call CanLup_DDS. We compared this genome to the Boxer (CanFam3.1) and German Shepherd dog (CanFam_GSD) assemblies and characterized lineage-specific and shared genetic variation ranging from single- to megabase pair-sized variants. We identified 21,483 dingo-specific and 16,595 domestic dog-specific homozygous structural variants mediating genic and putative regulatory changes. Comparisons between the dingo and domestic dog builds detected unique inversions on Chromosome 16, structural variations in genes linked with starch metabolism, and seven differentially methylated genes. To experimentally assess genomic differences 17 dingoes and 15 German Shepherd dogs were fed parallel diets for 14 days. In dingoes, low AMY2B copy number and serum amylase levels are linked with high cholesterol and LDL levels. Gut microbiome analyses revealed enrichment of the family Clostridiaceae, which can utilize complex resistant starch, while scat metabolome studies identified high phenylethyl alcohol concentrations that we posit are linked with territory marking. Our study provides compelling genomic, microbiome, and metabolomic links showing the dingo has distinct physiology from domestic breed dogs with a unique role in the ecosystem.

genomics↗

bioRxiv2020

Chromosome-length genome assembly and structural variations of the primal Basenji dog (Canis lupus familiaris) genome

BackgroundBasenjis are considered an ancient dog breed of central African origins that still live and hunt with tribesmen in the African Congo. Nicknamed the barkless dog, Basenjis possess unique phylogeny, geographical origins and traits, making their genome structure of great interest. The increasing number of available canid reference genomes allows us to examine the impact the choice of reference genome makes with regard to reference genome quality and breed relatedness. ResultsHere, we report two high quality de novo Basenji genome assemblies: a female, China (CanFam_Bas), and a male, Wags. We conduct pairwise comparisons and report structural variations between assembled genomes of three dog breeds: Basenji (CanFam_Bas), Boxer (CanFam3.1) and German Shepherd Dog (GSD) (CanFam_GSD). CanFam_Bas is superior to CanFam3.1 in terms of genome contiguity and comparable overall to the high quality CanFam_GSD assembly. By aligning short read data from 58 representative dog breeds to three reference genomes, we demonstrate how the choice of reference genome significantly impacts both read mapping and variant detection. ConclusionsThe growing number of high-quality canid reference genomes means the choice of reference genome is an increasingly critical decision in subsequent canid variant analyses. The basal position of the Basenji makes it suitable for variant analysis for targeted applications of specific dog breeds. However, we believe more comprehensive analyses across the entire family of canids is more suited to a pangenome approach. Collectively this work highlights the importance the choice of reference genome makes in all variation studies.

genomics↗

bioRxiv2020

D614G mutation alters SARS-CoV-2 spike conformational dynamics and protease cleavage susceptibility at the S1/S2 junction

The SARS-CoV-2 spike (S) protein is the target of vaccine design efforts to end the COVID-19 pandemic. Despite a low mutation rate, isolates with the D614G substitution in the S protein appeared early during the pandemic, and are now the dominant form worldwide. Here, we analyze the D614G mutation in the context of a soluble S ectodomain construct. Cryo-EM structures, antigenicity and proteolysis experiments suggest altered conformational dynamics resulting in enhanced furin cleavage efficiency of the G614 variant. Furthermore, furin cleavage altered the conformational dynamics of the Receptor Binding Domains (RBD) in the G614 S ectodomain, demonstrating an allosteric effect on the RBD dynamics triggered by changes in the SD2 region, that harbors residue 614 and the furin cleavage site. Our results elucidate SARS-CoV-2 spike conformational dynamics and allostery, and have implications for vaccine design. HighlightsO_LISARS-CoV-2 S ectodomains with or without the K986P, V987P mutations have similar structures, antigenicity and stability. C_LIO_LIThe D614G mutation alters S protein conformational dynamics. C_LIO_LID614G enhances protease cleavage susceptibility at the S protein furin cleavage site. C_LIO_LICryo-EM structures reveal allosteric effect of changes at the S1/S2 junction on RBD dynamics. C_LI

biophysics↗

bioRxiv2020

Cold sensitivity of the SARS-CoV-2 spike ectodomain

The SARS-CoV-2 spike (S) protein, a primary target for COVID-19 vaccine development, presents its Receptor Binding Domain in two conformations: receptor-accessible "up" or receptor-inaccessible "down" conformations. Here, we report that the commonly used stabilized S ectodomain construct "2P" is sensitive to cold temperature, and that this cold sensitivity is resolved in a "down" state stabilized spike. Our results will impact structural, functional and vaccine studies that use the SARS-CoV-2 S ectodomain.

biochemistry↗

bioRxiv2020

A glycan cluster on the SARS-CoV-2 spike ectodomain is recognized by Fab-dimerized glycan-reactive antibodies

SummaryThe COVID-19 pandemic caused by SARS-CoV-2 has escalated into a global crisis. The spike (S) protein that mediates cell entry and membrane fusion is the current focus of vaccine and therapeutic antibody development efforts. The S protein, like many other viral fusion proteins such as HIV-1 envelope (Env) and influenza hemagglutinin, is glycosylated with both complex and high mannose glycans. Here we demonstrate binding to the SARS-CoV-2 S protein by a category of Fab-dimerized glycan-reactive (FDG) HIV-1-induced broadly neutralizing antibodies (bnAbs). A 3.1 Å resolution cryo-EM structure of the S protein ectodomain bound to glycan-dependent HIV-1 bnAb 2G12 revealed a quaternary glycan epitope on the spike S2 domain involving multiple protomers. These data reveal a new epitope on the SARS-CoV-2 spike that can be targeted for vaccine design.HighlightsFab-dimerized, glycan-reactive (FDG) HIV-1 bnAbs cross-react with SARS-CoV-2 spike.3.1 Å resolution cryo-EM structure reveals quaternary S2 epitope for HIV-1 bnAb 2G12.2G12 targets glycans, at positions 709, 717 and 801, in the SARS-CoV-2 spike.Our studies suggest a common epitope for FDG antibodies centered around glycan 709.Competing Interest StatementThe authors have declared no competing interest.View Full Text

immunology↗

bioRxiv2020

Fab-dimerized glycan-reactive antibodies neutralize HIV and are prevalent in humans and rhesus macaques

SummaryThe HIV-1 envelope (Env) is comprised by mass of over 50% glycans. A goal of HIV-1 vaccine development is the induction of Env glycan-reactive broadly neutralizing antibodies (bnAbs). The 2G12 bnAb recognizes an Env glycan cluster using a unique variable heavy (VH) domain-swapped conformation that results in fragment antigen-binding (Fab) dimerization. Here we describe Fab-dimerized glycan (FDG)-reactive antibodies without VH-swapped domains from simian-human immunodeficiency virus (SHIV)-infected macaques that neutralized heterologous HIV-1 isolates. FDG precursors were boosted by vaccination in macaques, and were present in HIV-1-naïve humans with an average estimated frequency of one per 340,000 B cells. These data demonstrate frequent HIV-1 Env glycan-reactive bnAb B cell precursors in macaques and humans and reveal a novel strategy for their induction by vaccination.HighlightsDiscovery of Fab-dimerized HIV-1 glycan-reactive antibodies with a non-domain-swapped architectureFab-dimerized antibodies neutralize heterologous HIV-1 isolates.Antibodies with this architecture can be elicited by vaccination in macaques.Fab-dimerized antibodies are found in HIV-1 naïve humans.Competing Interest StatementThe authors have declared no competing interest.View Full Text

immunology↗

bioRxiv2020

Glycans on the SARS-CoV-2 Spike Control the Receptor Binding Domain Conformation

The glycan shield of the beta-coronavirus (β-CoV) Spike (S) glycoprotein provides protection from host immune responses, acting as a steric block to potentially neutralizing antibody responses. The conformationally dynamic S-protein is the primary immunogenic target of vaccine design owing to its role in host-cell fusion, displaying multiple receptor binding domain (RBD) ‘up’ and ‘down’ state configurations. Here, we investigated the potential for RBD adjacent, N-terminal domain (NTD) glycans to influence the conformational equilibrium of these RBD states. Using a combination of antigenic screens and high-resolution cryo-EM structure determination, we show that an N-glycan deletion at position 234 results in a dramatically reduced population of the ‘up’ state RBD position. Conversely, glycan deletion at position N165 results in a discernable increase in ‘up’ state RBDs. This indicates the glycan shield acts not only as a passive hinderance to antibody meditated immunity but also as a conformational control element. Together, our results demonstrate this highly dynamic conformational machine is responsive to glycan modification with implications in viral escape and vaccine design.Competing Interest StatementThe authors have declared no competing interest.View Full Text

biophysics↗

bioRxiv2020

Controlling the SARS-CoV-2 Spike Glycoprotein Conformation

The coronavirus (CoV) viral host cell fusion spike (S) protein is the primary immunogenic target for virus neutralization and the current focus of many vaccine design efforts. The highly flexible S-protein, with its mobile domains, presents a moving target to the immune system. Here, to better understand S-protein mobility, we implemented a structure-based vector analysis of available {beta}-CoV S-protein structures. We found that despite overall similarity in domain organization, different {beta}-CoV strains display distinct S-protein configurations. Based on this analysis, we developed two soluble ectodomain constructs in which the highly immunogenic and mobile receptor binding domain (RBD) is locked in either the all-RBDs down position or is induced to display a previously unobserved in SARS-CoV-2 2-RBDs up configuration. These results demonstrate that the conformation of the S-protein can be controlled via rational design and provide a framework for the development of engineered coronavirus spike proteins for vaccine applications.

immunology↗

bioRxiv2020

Sensory and motor electrophysiological mapping of the cerebellum in humans.

Damage to the cerebellum during posterior fossa surgery can lead to ataxia and in paediatric cases, the risk of cerebellar mutism syndrome. Animal electrophysiological and human imaging studies have shown compartmentalisation of sensorimotor and cognitive functions within the cerebellum. In the present study, electrophysiological monitoring of sensory and motor pathways was carried out to assess the location of limb sensorimotor representation within the human cerebellum, as a potential approach for real time assessment of neurophysiological integrity to reduce the incidence of cerebellar surgical morbidities. Thirteen adult and paediatric patients undergoing posterior fossa surgery were recruited. For sensory mapping (n=8), electrical stimulation was applied to the median nerves, the posterior tibial nerves, or proximal and distal limb muscles and evoked field potential responses were sought on the cerebellar surface. For motor mapping (n=5), electrical stimulation was applied to the surface of the cerebellum and evoked EMG responses were sought in facial and limb muscles. Evoked potentials on the cerebellar surface were found in two patients (25% of cases). In one patient, the evoked response was located on the surface of the right inferior posterior cerebellum in response to stimulation of the right leg. In the second patient, stimulation of the extensor digitorum muscle in the left forearm evoked a response on the surface of the left inferior posterior lobe. In the motor mapping cases no evoked EMG responses could be found. Intraoperative electrophysiological mapping, therefore, indicates it is possible to record evoked potentials on the surface of the human cerebellum in response to peripheral stimulation.

neuroscience↗