Biology subjects

Chen, R. E.

Publications and source records attributed to Chen, R. E..

11 recordsLinked to original sources

bioRxiv2021

Ultrapotent miniproteins targeting the receptor-binding domain protect against SARS-CoV-2 infection and disease in mice

Despite the introduction of public health measures and spike protein-based vaccines to mitigate the COVID-19 pandemic, SARS-CoV-2 infections and deaths continue to rise. Previously, we used a structural design approach to develop picomolar range miniproteins targeting the SARS-CoV-2 receptor binding domain. Here, we investigated the capacity of modified versions of one lead binder, LCB1, to protect against SARS-CoV-2-mediated lung disease in human ACE2-expressing transgenic mice. Systemic administration of LCB1-Fc reduced viral burden, diminished immune cell infiltration and inflammation, and completely prevented lung disease and pathology. A single intranasal dose of LCB1v1.3 reduced SARS-CoV-2 infection in the lung even when given as many as five days before or two days after virus inoculation. Importantly, LCB1v1.3 protected in vivo against a historical strain (WA1/2020), an emerging B.1.1.7 strain, and a strain encoding key E484K and N501Y spike protein substitutions. These data support development of LCB1v1.3 for prevention or treatment of SARS-CoV-2 infection.

microbiology↗

bioRxiv2020

Human neutralizing antibodies against SARS-CoV-2 require intact Fc effector functions and monocytes for optimal therapeutic protection

SARS-CoV-2 has caused the global COVID-19 pandemic. Although passively delivered neutralizing antibodies against SARS-CoV-2 show promise in clinical trials, their mechanism of action in vivo is incompletely understood. Here, we define correlates of protection of neutralizing human monoclonal antibodies (mAbs) in SARS-CoV-2-infected animals. Whereas Fc effector functions are dispensable when representative neutralizing mAbs are administered as prophylaxis, they are required for optimal protection as therapy. When given after infection, intact mAbs reduce SARS-CoV-2 burden and lung disease in mice and hamsters better than loss-of-function Fc variant mAbs. Fc engagement of neutralizing antibodies mitigates inflammation and improves respiratory mechanics, and transcriptional profiling suggests these phenotypes are associated with diminished innate immune signaling and preserved tissue repair. Immune cell depletions establish that neutralizing mAbs require monocytes for therapeutic efficacy. Thus, potently neutralizing mAbs require Fc effector functions for maximal therapeutic benefit during therapy to modulate protective immune responses and mitigate lung disease.

immunology↗

bioRxiv2020

Cross-reactive coronavirus antibodies with diverse epitope specificities and extra-neutralization functions

The continual emergence of novel coronavirus (CoV) strains, like SARS-CoV-2, highlights the critical need for broadly reactive therapeutics and vaccines against this family of viruses. Coronavirus spike (S) proteins share common structural motifs that could be vulnerable to cross-reactive antibody responses. To study this phenomenon in human coronavirus infection, we applied a high-throughput sequencing method called LIBRA-seq (Linking B cell receptor to antigen specificity through sequencing) to a SARS-CoV-1 convalescent donor sample. We identified and characterized a panel of six monoclonal antibodies that cross-reacted with S proteins from the highly pathogenic SARS-CoV-1 and SARS-CoV-2 and demonstrated a spectrum of reactivity against other coronaviruses. Epitope mapping revealed that these antibodies recognized multiple epitopes on SARS-CoV-2 S, including the receptor binding domain (RBD), N-terminal domain (NTD), and S2 subunit. Functional characterization demonstrated that the antibodies mediated a variety of Fc effector functions in vitro and mitigated pathological burden in vivo. The identification of cross-reactive epitopes recognized by functional antibodies expands the repertoire of targets for pan-coronavirus vaccine design strategies that may be useful for preventing potential future coronavirus outbreaks.

immunology↗

bioRxiv2020

Landscape analysis of escape variants identifies SARS-CoV-2 spike mutations that attenuate monoclonal and serum antibody neutralization

Although neutralizing antibodies against the SARS-CoV-2 spike (S) protein are a goal of COVID-19 vaccines and have received emergency use authorization as therapeutics, viral escape mutants could compromise their efficacy. To define the immune-selected mutational landscape in S protein, we used a VSV-eGFP-SARS-CoV-2-S chimeric virus and 19 neutralizing monoclonal antibodies (mAbs) against the receptor-binding domain (RBD) to generate 50 different escape mutants. The variants were mapped onto the RBD structure and evaluated for cross-resistance to mAbs and convalescent human sera. Each mAb had a unique resistance profile, although many shared residues within an epitope. Some variants (e.g., S477N) were resistant to neutralization by multiple mAbs, whereas others (e.g., E484K) escaped neutralization by convalescent sera, suggesting some humans induce a narrow repertoire of neutralizing antibodies. Comparing the antibody-mediated mutational landscape in S with sequence variation in circulating SARS-CoV-2, we define substitutions that may attenuate neutralizing immune responses in some humans.

microbiology↗

bioRxiv2020

De novo design of picomolar SARS-CoV-2 miniprotein inhibitors

We used two approaches to design proteins with shape and chemical complementarity to the receptor binding domain (RBD) of SARS-CoV-2 Spike protein near the binding site for the human ACE2 receptor. Scaffolds were built around an ACE2 helix that interacts with the RBD, or de novo designed scaffolds were docked against the RBD to identify new binding modes. In both cases, designed sequences were optimized first in silico and then experimentally for target binding, folding and stability. Nine designs bound the RBD with affinities ranging from 100pM to 10nM, and blocked bona fide SARS-CoV-2 infection of Vero E6 cells with IC50 values ranging from 35 pM to 35 nM; the most potent of these -- 56 and 64 residue hyperstable proteins made using the second approach -- are roughly six times more potent on a per mass basis (IC50 ~ 0.23 ng/ml) than the best monoclonal antibodies reported thus far. Cryo-electron microscopy structures of the SARS-CoV-2 spike ectodomain trimer in complex with the two most potent minibinders show that the structures of the designs and their binding interactions with the RBD are nearly identical to the computational models, and that all three RBDs in a single Spike protein can be engaged simultaneously. These hyperstable minibinders provide promising starting points for new SARS-CoV-2 therapeutics, and illustrate the power of computational protein design for rapidly generating potential therapeutic candidates against pandemic threats.

synthetic biology↗

bioRxiv2020

A single intranasal dose of chimpanzee adenovirus-vectored vaccine confers sterilizing immunity against SARS-CoV-2 infection

The Coronavirus Disease 2019 pandemic has made deployment of an effective vaccine a global health priority. We evaluated the protective activity of a chimpanzee adenovirus-vectored vaccine encoding a pre-fusion stabilized spike protein (ChAd-SARS-CoV-2-S) in challenge studies with Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and mice expressing the human angiotensin-converting enzyme 2 receptor. Intramuscular dosing of ChAd-SARS-CoV-2-S induces robust systemic humoral and cell-mediated immune responses and protects against lung infection, inflammation, and pathology but does not confer sterilizing immunity, as evidenced by detection of viral RNA and induction of anti-nucleoprotein antibodies after SARS-CoV-2 challenge. In contrast, a single intranasal dose of ChAd-SARS-CoV-2-S induces high levels of systemic and mucosal IgA and T cell responses, completely prevents SARS-CoV-2 infection in the upper and lower respiratory tracts, and likely confers sterilizing immunity in most animals. Intranasal administration of ChAd-SARS-CoV-2-S is a candidate for preventing SARS-CoV-2 infection and transmission, and curtailing pandemic spread.

microbiology↗

bioRxiv2020

Association between neutralizing antibodies to SARS-CoV-2 and commercial serological assays

Introduction Commercially available SARS-CoV-2 serological assays based on different viral antigens have been approved for the qualitative determination of anti-SARS-CoV-2 antibodies. However, there is limited published data associating the results from commercial assays with neutralizing antibodies.Methods 67 specimens from 48 patients with PCR-confirmed COVID-19 and a positive result by the Roche Elecsys SARS-CoV-2, Abbott SARS-CoV-2 IgG, or EUROIMMUN SARS-CoV-2 IgG assays and 5 control specimens were analyzed for the presence of neutralizing antibodies to SARS-CoV-2. Correlation, concordance, positive percent agreement (PPA), and negative percent agreement (NPA) were calculated at several cutoffs. Results were compared in patients categorized by clinical outcomes.Results The correlation between SARS-CoV-2 neutralizing titer (EC50) and the Roche, Abbott, and EUROIMMUN assays was 0.29, 0.47, and 0.46 respectively. At an EC50 of 1:32, the concordance kappa with Roche was 0.49 (95% CI; 0.23-0.75), with Abbott was 0.52 (0.28-0.77), and with EUROIMMUN was 0.61 (0.4-0.82). At the same neutralizing titer, the PPA and NPA for the Roche was 100% (94-100) & 56% (30-80); Abbott was 96% (88-99) & 69% (44-86); and EUROIMMUN was 91% (80-96) & 81% (57-93) for distinguishing neutralizing antibodies. Patients who died, were intubated, or had a cardiac injury from COVID-19 infection had significantly higher neutralizing titers relative to those with mild symptoms.Conclusion COVID-19 patients generate an antibody response to multiple viral proteins such that the calibrator ratios on the Roche, Abbott, and EUROIMMUN assays are all associated with SARS-CoV-2 neutralization. Nevertheless, commercial serological assays have poor NPA for SARS-CoV-2 neutralization, making them imperfect proxies for neutralization.Competing Interest StatementThe authors have declared no competing interest.View Full Text

immunology↗

bioRxiv2020

Potently neutralizing human antibodies that block SARS-CoV-2 receptor binding and protect animals

The COVID-19 pandemic is a major threat to global health for which there are only limited medical countermeasures, and we lack a thorough understanding of mechanisms of humoral immunity1,2. From a panel of monoclonal antibodies (mAbs) targeting the spike (S) glycoprotein isolated from the B cells of infected subjects, we identified several mAbs that exhibited potent neutralizing activity with IC50 values as low as 0.9 or 15 ng/mL in pseudovirus or wild-type (wt) SARS-CoV-2 neutralization tests, respectively. The most potent mAbs fully block the receptor-binding domain of S (SRBD) from interacting with human ACE2. Competition-binding, structural, and functional studies allowed clustering of the mAbs into defined classes recognizing distinct epitopes within major antigenic sites on the SRBD. Electron microscopy studies revealed that these mAbs recognize distinct conformational states of trimeric S protein. Potent neutralizing mAbs recognizing unique sites, COV2-2196 and COV2-2130, bound simultaneously to S and synergistically neutralized authentic SARS-CoV-2 virus. In two murine models of SARS-CoV-2 infection, passive transfer of either COV2-2916 or COV2-2130 alone or a combination of both mAbs protected mice from severe weight loss and reduced viral burden and inflammation in the lung. These results identify protective epitopes on the SRBD and provide a structure-based framework for rational vaccine design and the selection of robust immunotherapeutic cocktails.

immunology↗

bioRxiv2020

Neutralizing antibody and soluble ACE2 inhibition of a replication-competent VSV-SARS-CoV-2 and a clinical isolate of SARS-CoV-2.

Antibody-based interventions against SARS-CoV-2 could limit morbidity, mortality, and possibly disrupt epidemic transmission. An anticipated correlate of such countermeasures is the level of neutralizing antibodies against the SARS-CoV-2 spike protein, yet there is no consensus as to which assay should be used for such measurements. Using an infectious molecular clone of vesicular stomatitis virus (VSV) that expresses eGFP as a marker of infection, we replaced the glycoprotein gene (G) with the spike protein of SARS-CoV-2 (VSV-eGFP-SARS-CoV-2) and developed a high-throughput imaging-based neutralization assay at biosafety level 2. We also developed a focus reduction neutralization test with a clinical isolate of SARS-CoV-2 at biosafety level 3. We compared the neutralizing activities of monoclonal and polyclonal antibody preparations, as well as ACE2-Fc soluble decoy protein in both assays and find an exceptionally high degree of concordance. The two assays will help define correlates of protection for antibody-based countermeasures including therapeutic antibodies, immune {gamma}-globulin or plasma preparations, and vaccines against SARS-CoV-2. Replication-competent VSV-eGFP-SARS-CoV-2 provides a rapid assay for testing inhibitors of SARS-CoV-2 mediated entry that can be performed in 7.5 hours under reduced biosafety containment.

microbiology↗

bioRxiv2020

Inhibition of PIKfyve kinase prevents infection by EBOV and SARS-CoV-2

Virus entry is a multistep process. It initiates when the virus attaches to the host cell and ends when the viral contents reach the cytosol. Genetically unrelated viruses can subvert analogous subcellular mechanisms and use similar trafficking pathways for successful entry. Antiviral strategies targeting early steps of infection are therefore appealing, particularly when the probability for successful interference through a common step is highest. We describe here potent inhibitory effects on content release and infection by chimeric VSV containing the envelope proteins of Zaire ebolavirus (VSV-ZEBOV) or SARS-CoV-2 (VSV-SARS-CoV-2) elicited by Apilimod and Vacuolin-1, small molecule inhibitors of the main endosomal Phosphatidylinositol-3-Phosphate/Phosphatidylinositol 5-Kinase, PIKfyve. We also describe potent inhibition of SARS-CoV-2 strain 2019-nCoV/USA-WA1/2020 by Apilimod. These results define new tools for studying the intracellular trafficking of pathogens elicited by inhibition of PIKfyve kinase and suggest the potential for targeting this kinase in developing small-molecule antivirals against SARS-CoV-2.

cell biology↗

bioRxiv2020

Structural and functional analysis of a potent sarbecovirus neutralizing antibody

SARS-CoV-2 is a newly emerged coronavirus responsible for the current COVID-19 pandemic that has resulted in more than one million infections and 73,000 deaths1,2. Vaccine and therapeutic discovery efforts are paramount to curb the pandemic spread of this zoonotic virus. The SARS-CoV-2 spike (S) glycoprotein promotes entry into host cells and is the main target of neutralizing antibodies. Here we describe multiple monoclonal antibodies targeting SARS-CoV-2 S identified from memory B cells of a SARS survivor infected in 2003. One antibody, named S309, potently neutralizes SARS-CoV-2 and SARS-CoV pseudoviruses as well as authentic SARS-CoV-2 by engaging the S receptor-binding domain. Using cryo-electron microscopy and binding assays, we show that S309 recognizes a glycan-containing epitope that is conserved within the sarbecovirus subgenus, without competing with receptor attachment. Antibody cocktails including S309 along with other antibodies identified here further enhanced SARS-CoV-2 neutralization and may limit the emergence of neutralization-escape mutants. These results pave the way for using S309 and S309-containing antibody cocktails for prophylaxis in individuals at high risk of exposure or as a post-exposure therapy to limit or treat severe disease.

immunology↗