Biology subjects

Crowe, J. E.

Publications and source records attributed to Crowe, J. E..

47 records · Page 3Linked to original sources

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

Convergence of a common solution to broad ebolavirus neutralization by glycan cap directed human antibodies

Antibodies that target the glycan cap epitope on ebolavirus glycoprotein (GP) are common in the adaptive response of survivors. A subset is known to be broadly neutralizing, but the details of their epitopes and basis for neutralization is not well-understood. Here we present cryo-electron microscopy (cryo-EM) structures of several glycan cap antibodies that variably synergize with GP base-binding antibodies. These structures describe a conserved site of vulnerability that anchors the mucin-like domains (MLD) to the glycan cap, which we name the MLD-anchor and cradle. Antibodies that bind to the MLD-cradle share common features, including the use of IGHV1-69 and IGHJ6 germline genes, which exploit hydrophobic residues and form beta-hairpin structures to mimic the MLD-anchor, disrupt MLD attachment, destabilize GP quaternary structure and block cleavage events required for receptor binding. Our results collectively provide a molecular basis for ebolavirus neutralization by broadly reactive glycan cap antibodies.

immunology↗

bioRxiv2020

Complete mapping of mutations to the SARS-CoV-2 spike receptor-binding domain that escape antibody recognition

Antibodies targeting the SARS-CoV-2 spike receptor-binding domain (RBD) are being developed as therapeutics and make a major contribution to the neutralizing antibody response elicited by infection. Here, we describe a deep mutational scanning method to map how all amino-acid mutations in the RBD affect antibody binding, and apply this method to 10 human monoclonal antibodies. The escape mutations cluster on several surfaces of the RBD that broadly correspond to structurally defined antibody epitopes. However, even antibodies targeting the same RBD surface often have distinct escape mutations. The complete escape maps predict which mutations are selected during viral growth in the presence of single antibodies, and enable us to design escape-resistant antibody cocktails-including cocktails of antibodies that compete for binding to the same surface of the RBD but have different escape mutations. Therefore, complete escape-mutation maps enable rational design of antibody therapeutics and assessment of the antigenic consequences of viral evolution.

microbiology↗

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

Integrated technology platform for accelerated discovery of antiviral antibody therapeutics

The emergence and reemergence of highly virulent viral pathogens with pandemic potential has created an urgent need for accelerated discovery of antiviral therapeutics. Antiviral human monoclonal (mAbs) are promising drug candidates to prevent or treat severe viral diseases, but the long timelines needed for discovery limits their rapid deployment and use. Here, we report the development of an integrated sequence of technologies incorporating advances in single-cell mRNA sequence analysis, bioinformatics, synthetic biology, and high-throughput functional analysis that allowed us to discover highly potent antiviral human mAbs and validate their activity in vivo at an unprecedented scale, speed, and efficiency. In a 78-day study, modeling deployment of a rapid response platform to an outbreak, we isolated >100 individual Zika virus (ZIKV) specific human mAbs, assessed their function, identified 29 broadly-neutralizing mAbs, and verified therapeutic potency of lead candidates with antibody-encoding mRNA formulation and/or IgG protein delivery in mice and nonhuman primates. Our work provides a roadmap for the rapid antibody discovery programs against viral pathogens of global concern.

immunology↗

bioRxiv2020

Rapid isolation and profiling of a diverse panel of human monoclonal antibodies targeting the SARS-CoV-2 spike protein

Antibodies are a principal determinant of immunity for most RNA viruses and have promise to reduce infection or disease during major epidemics. The novel coronavirus SARS-CoV-2 has caused a global pandemic with millions of infections and hundreds of thousands of deaths to date1,2. In response, we used a rapid antibody discovery platform to isolate hundreds of human monoclonal antibodies (mAbs) against the SARS-CoV-2 spike (S) protein. We stratify these mAbs into five major classes based on their reactivity to subdomains of S protein as well as their cross-reactivity to SARS-CoV. Many of these mAbs inhibit infection of authentic SARS-CoV-2 virus, with most neutralizing mAbs recognizing the receptor-binding domain (RBD) of S. This work defines sites of vulnerability on SARS-CoV-2 S and demonstrates the speed and robustness of new antibody discovery methodologies.

immunology↗

bioRxiv2019

Multi-State Design of Flexible Proteins Predicts Sequences Optimal for Conformational Change

Computational protein design of an ensemble of conformations for one protein - i.e., multi-state design - determines the side chain identity by optimizing the energetic contributions of that side chain in each of the backbone conformations. Sampling the resulting large sequence-structure search space limits the number of conformations and the size of proteins in multi-state design algorithms. Here, we demonstrated that the REstrained CONvergence (RECON) algorithm can simultaneously evaluate the sequence of large proteins that undergo substantial conformational changes, such as viral surface glycoproteins. Simultaneous optimization of side chain conformations across all conformations resulted in an increase of 30% to 40% in sequence conservation when compared to single-state designs. More importantly, the sampled sequence space of RECON designs resembled the evolutionary sequence space of functional proteins. This finding was especially true for sequence positions that require substantial changes in their local environment across an ensemble of conformations. To quantify this rewiring of contacts at a certain position in sequence and structure, we introduced a new metric designated contact proximity deviation that enumerates contact map changes. This measure allows mapping of global conformational changes into local side chain proximity adjustments, a property not captured by traditional global similarity metrics such as RMSD or local similarity metrics such as changes in {varphi} and {psi} angles.\n\nAuthor SummaryMulti-state design can be used to engineer proteins that need to exist in multiple conformations or that bind to multiple partner molecules. In essence, multi-state design selects a compromise of protein sequences that allow for an ensemble of protein conformations, or states, associated with a particular biological function. In this paper, we used the REstrained CONvergence (RECON) algorithm with Rosetta to show that multi-state design of flexible proteins predicts sequences optimal for conformational change, mimicking mutation preferences sampled in evolution. Modeling optimal local side chain physicochemical environments within an ensemble selected significantly more native-like sequences than selections performed when all conformations states are designed independently. This outcome was particularly true for amino acids whose local side chain environment change between conformations. To quantify such contact map changes, we introduced a novel metric to show that sequence conservation is dependent on protein flexibility, i.e., changes in local side chain environments between stated limit the space of tolerated mutations. Additionally, such positions in sequence and structure are more likely to be energetically frustrated, at least in some states. Importantly, we showed that multi-state design over an ensemble of conformations (space) can explore evolutionary tolerated sequence space (time), thus enabling RECON to not only design proteins that require multiple states for function but also predict mutations that might be tolerated in native proteins but have not yet been explored by evolution. The latter aspect can be important to anticipate escape mutations, for example in pathogens or oncoproteins.

biophysics↗

bioRxiv2019

Peptide Arrays of Three Collections of Human Sera from Patients Infected with Mosquito-Borne Viruses

Global outbreaks and epidemics caused by emerging or re-emerging mosquito-borne viruses are becoming more common. These viruses belong to multiple genera including Flavivirus and Alphavirus and often cause non-specific or asymptomatic infection, which can confound viral prevalence studies. In addition, many acute phase diagnostic tests rely on the detection of viral components such as RNA or antigen. Standard serological tests are often not reliable for diagnosis after the acute phase due to cross-reactivity among viruses (e.g. flaviviruses). In order to contribute to development efforts for mosquito-borne serodiagnostics, we incubated 137 human sera on individual custom peptide arrays that consisted of over 866 unique peptides in quadruplicate. Our bioinformatics workflow to analyze these data incorporated machine learning, statistics, and B-cell epitope prediction. The unprocessed array data can be useful in separate meta-analyses that can be applicable to diverse efforts including the development of new pan-flavivirus antibodies, more accurate epitope mapping, and vaccine development against these viral pathogens.

microbiology↗

bioRxiv2019

Broad Dengue Neutralization in Mosquitoes Expressing an Engineered Antibody

With dengue virus (DENV) becoming endemic in tropical and subtropical regions worldwide, there is a pressing global demand for effective strategies to control the mosquitoes that spread this disease. Recent advances in genetic engineering technologies have made it possible to create mosquitoes with reduced vector competence, limiting their ability to acquire and transmit pathogens. Here we describe the development of Aedes aegypti mosquitoes synthetically engineered to impede vector competence to DENV. These mosquitoes express a gene encoding an engineered single-chain variable fragment derived from a broadly neutralizing DENV human monoclonal antibody and have significantly reduced viral infection, dissemination, and transmission rates for all four major antigenically distinct DENV serotypes. Importantly, this is the first engineered approach that targets all DENV serotypes, which is crucial for effective disease suppression. These results provide a compelling route for developing effective genetic-based DENV control strategies, which could be extended to curtail other arboviruses.\n\nSignificance StatementHere we describe the development of Ae. aegypti mosquitoes synthetically engineered to impede vector competence to multiple serotypes of DENV. Given the global significance of DENV, these mosquitoes may be used to curtail Dengue transmission by this potent vector.

bioengineering↗

bioRxiv2018

Potent anti-influenza H7 human monoclonal antibody induces separation of hemagglutinin receptor binding head domains

Seasonal influenza virus infections can cause significant morbidity and mortality, but the threat from emergence of a new pandemic influenza strain might have potentially even more devastating consequences. As such, there is intense interest in isolating and characterizing potent neutralizing antibodies that target the hemagglutinin (HA) viral surface glycoprotein. Here, we use cryo-electron microscopy to decipher the mechanism of action of a potent HA head-directed monoclonal antibody bound to an influenza H7 HA. The epitope of the antibody is not solvent accessible in the compact, pre-fusion conformation that typifies all HA structures to date. Instead, the antibody binds between HA head protomers to an epitope that must be partly or transiently exposed in the pre-fusion conformation. The \"breathing\" of the HA protomers is implied by the exposure of this epitope, which is consistent with metastability of class I fusion proteins. This structure likely therefore represents an early structural intermediate in the viral fusion process. Understanding the extent of transient exposure of conserved neutralizing epitopes also may lead to new opportunities to combat influenza that have not been appreciated previously.\n\nAuthor SummaryA transiently exposed epitope on influenza H7 hemagglutinin represents a new target for neutralizing antibodies.

immunology↗