Biology subjects

Hsieh, C.-L.

Publications and source records attributed to Hsieh, C.-L..

28 records · Page 2Linked to original sources

bioRxiv2021

Early cross-coronavirus reactive signatures of protective humoral immunity against COVID-19

The introduction of vaccines has inspired new 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 COVID-19, thus we profiled the earliest humoral signatures in a large cohort of severe and asymptomatic COVID-19 individuals. While a SARS-CoV-2-specific immune response evolved rapidly in survivors of COVID-19, non-survivors exhibited blunted and delayed humoral immune evolution, particularly with respect to S2-specific antibody evolution. Given the conservation of S2 across {beta}-coronaviruses, we found the early development of SARS-CoV-2-specific immunity occurred in tandem with pre-existing common {beta}-coronavirus OC43 humoral immunity in survivors, which was selectively also expanded in individuals that develop paucisymptomatic infection. These data point to the importance of cross-coronavirus immunity as a correlate of protection against COVID-19.

immunology↗

bioRxiv2021

Elicitation of broadly protective sarbecovirus immunity by receptor-binding domain nanoparticle vaccines

Understanding the ability of SARS-CoV-2 vaccine-elicited antibodies to neutralize and protect against emerging variants of concern and other sarbecoviruses is key for guiding vaccine development decisions and public health policies. We show that a clinical stage multivalent SARS-CoV-2 receptor-binding domain nanoparticle vaccine (SARS-CoV-2 RBD-NP) protects mice from SARS-CoV-2-induced disease after a single shot, indicating that the vaccine could allow dose-sparing. SARS-CoV-2 RBD-NP elicits high antibody titers in two non-human primate (NHP) models against multiple distinct RBD antigenic sites known to be recognized by neutralizing antibodies. We benchmarked NHP serum neutralizing activity elicited by RBD-NP against a lead prefusion-stabilized SARS-CoV-2 spike immunogen using a panel of single-residue spike mutants detected in clinical isolates as well as the B.1.1.7 and B.1.351 variants of concern. Polyclonal antibodies elicited by both vaccines are resilient to most RBD mutations tested, but the E484K substitution has similar negative consequences for neutralization, and exhibit modest but comparable neutralization breadth against distantly related sarbecoviruses. We demonstrate that mosaic and cocktail sarbecovirus RBD-NPs elicit broad sarbecovirus neutralizing activity, including against the SARS-CoV-2 B.1.351 variant, and protect mice against severe SARS-CoV challenge even in the absence of the SARS-CoV RBD in the vaccine. This study provides proof of principle that sarbecovirus RBD-NPs induce heterotypic protection and enables advancement of broadly protective sarbecovirus vaccines to the clinic.

immunology↗

bioRxiv2021

Adjuvanting a subunit SARS-CoV-2 nanoparticle vaccine to induce protective immunity in non-human primates

The development of a portfolio of SARS-CoV-2 vaccines to vaccinate the global population remains an urgent public health imperative. Here, we demonstrate the capacity of a subunit vaccine under clinical development, comprising the SARS-CoV-2 Spike protein receptor binding domain displayed on a two-component protein nanoparticle (RBD-NP), to stimulate robust and durable neutralizing antibody (nAb) responses and protection against SARS-CoV-2 in non-human primates. We evaluated five different adjuvants combined with RBD-NP including Essai O/W 1849101, a squalene-in-water emulsion; AS03, an alpha-tocopherol-containing squalene-based oil-in-water emulsion used in pandemic influenza vaccines; AS37, a TLR-7 agonist adsorbed to Alum; CpG 1018-Alum (CpG-Alum), a TLR-9 agonist formulated in Alum; or Alum, the most widely used adjuvant. All five adjuvants induced substantial nAb and CD4 T cell responses after two consecutive immunizations. Durable nAb responses were evaluated for RBD-NP/AS03 immunization and the live-virus nAb response was durably maintained up to 154 days post-vaccination. AS03, CpG-Alum, AS37 and Alum groups conferred significant protection against SARS-CoV-2 infection in the pharynges, nares and in the bronchoalveolar lavage. The nAb titers were highly correlated with protection against infection. Furthermore, RBD-NP when used in conjunction with AS03 was as potent as the prefusion stabilized Spike immunogen, HexaPro. Taken together, these data highlight the efficacy of the RBD-NP formulated with clinically relevant adjuvants in promoting robust immunity against SARS-CoV-2 in non-human primates.

immunology↗

bioRxiv2021

Identification of a conserved neutralizing epitope present on spike proteins from highly pathogenic coronaviruses

Three pathogenic human coronaviruses have emerged within the last 20 years, with SARS-CoV-2 causing a global pandemic. Although therapeutic antibodies targeting the SARS-CoV-2 spike currently focus on the poorly conserved receptor-binding domain, targeting essential neutralizing epitopes on the more conserved S2 domain may provide broader protection. We report an antibody binding an epitope conserved in the pre-fusion core of MERS-CoV, SARS-CoV and SARS-CoV-2 spike S2 domains. Antibody 3A3 binds a conformational epitope with ~2.5 nM affinity and neutralizes spike from SARS-CoV, SARS-CoV-2 and variants of concern in in vitro pseudovirus assays. Hydrogen-deuterium exchange mass spectrometry identified residues 980-1006 in the flexible hinge region at the S2 apex as the 3A3 epitope, suggesting 3A3 prevents the S2 conformational rearrangements required for conversion to the spike post-fusion state and virus-host cell fusion. This work defines a conserved vulnerable site on the SARS-CoV-2 S2 domain and guides the design of pan-protective spike immunogens.

biochemistry↗

bioRxiv2021

D614G Substitution of SARS-CoV-2 Spike Protein Increases Syncytium Formation and Viral Transmission via Enhanced Furin-mediated Spike Cleavage

Since the D614G substitution in the spike (S) of SARS-CoV-2 emerged, the variant strain underwent rapid expansion to become the most abundant strain worldwide. Therefore, this substitution may provide an advantage of viral spreading. To explore the mechanism, we analyzed 18 viral isolates containing S proteins with either G614 or D614. Both the virus titer and syncytial phenotype were significantly increased in S-G614 than in S-D614 isolates. We further showed increased cleavage of S at the furin substrate site, a key event that promotes syncytium, in S-G614 isolates. These functions of the D614G substitution were validated in cells expressing S protein. The effect on syncytium was abolished by furin inhibitor treatment and mutation of the furin-cleavage site, suggesting its dependence on cleavage by furin. Our study provides a mechanistic explanation for the increased transmissibility of S-G614 containing SARS-CoV-2 through enhanced furin-mediated S cleavage, which increases membrane fusion and virus infectivity.

microbiology↗

bioRxiv2020

Structure-based Design of Prefusion-stabilized SARS-CoV-2 Spikes

The COVID-19 pandemic caused by the novel coronavirus SARS-CoV-2 has led to accelerated efforts to develop therapeutics, diagnostics, and vaccines to mitigate this public health emergency. A key target of these efforts is the spike (S) protein, a large trimeric class I fusion protein that is metastable and difficult to produce recombinantly in large quantities. Here, we designed and expressed over 100 structure-guided spike variants based upon a previously determined cryo-EM structure of the prefusion SARS-CoV-2 spike. Biochemical, biophysical and structural characterization of these variants identified numerous individual substitutions that increased protein yields and stability. The best variant, HexaPro, has six beneficial proline substitutions leading to [~]10-fold higher expression than its parental construct and is able to withstand heat stress, storage at room temperature, and multiple freeze-thaws. A 3.2 [A]-resolution cryo-EM structure of HexaPro confirmed that it retains the prefusion spike conformation. High-yield production of a stabilized prefusion spike protein will accelerate the development of vaccines and serological diagnostics for SARS-CoV-2.

microbiology↗

bioRxiv2020

Broad sarbecovirus neutralizing antibodies define a key site of vulnerability on the SARS-CoV-2 spike protein

Broadly protective vaccines against known and pre-emergent coronaviruses are urgently needed. Critical to their development is a deeper understanding of cross-neutralizing antibody responses induced by natural human coronavirus (HCoV) infections. Here, we mined the memory B cell repertoire of a convalescent SARS donor and identified 200 SARS-CoV-2 binding antibodies that target multiple conserved sites on the spike (S) protein. A large proportion of the antibodies display high levels of somatic hypermutation and cross-react with circulating HCoVs, suggesting recall of pre-existing memory B cells (MBCs) elicited by prior HCoV infections. Several antibodies potently cross-neutralize SARS-CoV, SARS-CoV-2, and the bat SARS-like virus WIV1 by blocking receptor attachment and inducing S1 shedding. These antibodies represent promising candidates for therapeutic intervention and reveal a new target for the rational design of pan-sarbecovirus vaccines.

immunology↗

bioRxiv2020

Cryo-EM Structure of the 2019-nCoV Spike in the Prefusion Conformation

The outbreak of a novel betacoronavirus (2019-nCov) represents a pandemic threat that has been declared a public health emergency of international concern. The CoV spike (S) glycoprotein is a key target for urgently needed vaccines, therapeutic antibodies, and diagnostics. To facilitate medical countermeasure (MCM) development we determined a 3.5 [A]-resolution cryo-EM structure of the 2019-nCoV S trimer in the prefusion conformation. The predominant state of the trimer has one of the three receptor-binding domains (RBDs) rotated up in a receptor-accessible conformation. We also show biophysical and structural evidence that the 2019-nCoV S binds ACE2 with higher affinity than SARS-CoV S. Additionally we tested several published SARS-CoV RBD-specific monoclonal antibodies and found that they do not have appreciable binding to nCoV-2019 S, suggesting antibody cross-reactivity may be limited between the two virus RBDs. The atomic-resolution structure of 2019-nCoV S should enable rapid development and evaluation of MCMs to address the ongoing public health crisis.

microbiology↗

bioRxiv2019

Characterization of Single Protein Dynamics in Cell Plasma Membrane Derived Polymer Cushioned Lipid Bilayers

Native cell membrane derived supported lipid bilayers (SLBs) are emerging platforms that have broad applications ranging from fundamental research to next-generation biosensors. Central to the success of the platform is proper accommodation of membrane proteins so that their dynamics and functions are preserved. Polymer cushions have been commonly employed to avoid direct contact of the bilayer membrane to the supporting substrate, and thus the mobility of transmembrane proteins is maintained. However, little is known about how the polymer cushion affects the absolute mobility of membrane molecules. Here, we characterized the dynamics of single membrane proteins in polymer-cushioned lipid bilayers derived from cell plasma membranes and investigated the effects of polymer length. Three membrane proteins of distinct structures, i.e., GPI-anchored protein, single-pass transmembrane protein CD98 heavy chain, and seven-pass transmembrane protein SSTR3, were fused with green fluorescence proteins (GFPs) and their dynamics were measured by fluorescence single-molecule tracking. An automated data acquisition was implemented to study the effects of PEG polymer length to protein dynamics with large statistics. Our data showed that increasing the PEG polymer length (molecular weight from 1,000 to 5,000) enhanced the mobile fraction of the membrane proteins. Moreover, the diffusion coefficients of transmembrane proteins were raised by increasing the polymer length, whereas the diffusion coefficient of GPI-anchored protein remained almost identical with different polymer lengths. Importantly, the diffusion coefficients of the three membrane proteins became identical (2.5 m2/s approximately) in the cushioned membrane with the longest polymer length (molecular weight of 5,000), indicating that the SLBs were fully suspended from the substrate by the polymer cushion at the microscopic length scale. Transient confinements were observed from all three proteins, and increasing the polymer length reduced the tendency of transient confinements. The measured dynamics of membrane proteins were found to be nearly unchanged after depletion of cholesterol, suggesting that the observed immobilization and transient confinement were not due to cholesterol-enriched membrane nanodomains (lipid rafts). Our single-molecule dynamics elucidate the biophysical properties of polymer cushioned plasma membrane bilayers that are potentially useful for future developments of membrane-based biosensors and analytical assays.

biophysics↗

bioRxiv2018

The Organization of Membranes Below 100 nm: Experimental Breakthroughs Occasion a Modeling Manifesto

New experimental techniques reveal the plasma membrane to be heterogeneous and \"scale-rich,\" from nanometers to microns, and from microseconds to seconds. This is critical information, as heterogeneous, scale-dependent transport governs the molecular encounters that underlie cellular signaling. The data are rich, and reaffirm the importance of the cortical cytoskeleton, protein aggregates, and lipidomic complexity to the statistics of molecular encounters. Moreover, the data demand simulation approaches with a particular set of features, hence the \"manifesto\". Together with the experimental data, simulations which satisfy these requirements hold the promise of a deeper understanding of membrane spatiotemporal organization. Several experimental breakthroughs are reviewed, the constraints that they place on simulations are discussed, and the status of simulation approaches which aim to meet them are detailed.

biophysics↗