Biology subjects

Cleveland, T. E.

Publications and source records attributed to Cleveland, T. E..

3 recordsLinked to original sources

bioRxiv2024

Glycoengineered recombinant alpha1-antitrypsin results in comparable in vitro and in vivo activities to human plasma-derived protein

Alpha-1-antitrypsin (A1AT) is a multifunctional, clinically important, high value therapeutic glycoprotein that can be used for the treatment of many diseases such as alpha-1-antitrypsin deficiency, diabetes, graft-versus-host-disease, cystic fibrosis and various viral infections. Currently, the only FDA-approved treatment for A1AT disorders is intravenous augmentation therapy with human plasma-derived A1AT. In addition to its limited supply, this approach poses a risk of infection transmission, since it uses therapeutic A1AT harvested from donors. To address these issues, we sought to generate recombinant human A1AT (rhA1AT) that is chemically and biologically indistinguishable from its plasma-derived counterpart using glycoengineered Chinese Hamster Ovary (geCHO-L) cells. By deleting nine key genes that are part of the CHO glycosylation machinery and expressing the human ST6GAL1 and A1AT genes, we obtained stable, high producing geCHO-L lines that produced rhA1AT having an identical glycoprofile to plasma-derived A1AT (pdA1AT). Additionally, the rhA1AT demonstrated in vitro activity and in vivo half-life comparable to commercial pdA1AT. Thus, we anticipate that this platform will help produce human-like recombinant plasma proteins, thereby providing a more sustainable and reliable source of therapeutics that are cost-effective and better-controlled with regard to purity, clinical safety and quality.

bioengineering↗

bioRxiv2023

Cross-reactive macaque antibodies targeting marburgvirus glycoprotein induced by multivalent immunization

We utilized B cells from a Rhesus macaque immunized with a multivalent prime-boost regimen of filovirus antigens to isolate a novel panel of marburgvirus glycoprotein (GP)-specific monoclonal antibodies (mAbs). A heterologous marburgvirus GP probe was used to sort for B cells with cross-marburgvirus reactive breadth. 33 mAbs belonging to 28 unique lineages were expressed and experimentally characterized. Antibody specificities were assessed by binding competition and overlapping pepscan analyses, and were found to map to a previously characterized protective region on GP2 and a cross-filovirus reactive region on GP1, among others. A third of the lineages targeted the predicted receptor binding region (RBR), including two lineages with potent Marburg pseudovirus neutralization that were structurally analyzed and confirmed to recognize this region. Our study describes the discovery and characterization of a diverse panel of antibodies against marburgvirus GP induced by multivalent immunization and provides candidate immunotherapeutics for further study and development. Author SummaryMarburgviruses were the first filoviruses characterized to emerge in humans in 1967, and have led to multiple outbreaks since then with average case fatality rates of [~]50%. Although a vaccine and monoclonal antibody countermeasures have been approved for clinical use against the related Ebola viruses, these are ineffective against marburgviruses or other filoviruses. As such, gaps exist in the clinical toolkit against filoviruses, in particular marburgviruses. Here, we isolated and characterized a novel panel of monoclonal antibodies directed against the marburgvirus surface glycoprotein from an immunized Rhesus macaque. We utilized an antibody isolation method that ensured broad antibody recognition across multiple marburgvirus isolates. Functional and structural analyses revealed that roughly half of the antibodies in the panel mapped to regions on the glycoprotein shown previously to protect from infection, including the receptor binding domain and a protective region on the membrane-anchoring subunit, while a quarter of the antibodies did not fall into any known binding competition group indicating potential novel specificities. Our study advances the understanding of marburgvirus glycoprotein antigenicity and furthers efforts to develop candidate antibody countermeasures against these lethal viruses.

immunology↗

bioRxiv2023

Variable Gain DNA Nanostructure Charge Amplifiers for Biosensing

Electronic measurements of engineered nanostructures comprised solely of DNA (DNA nanostructures) enable new signal conditioning modalities in biosensing. Here, we demonstrate how DNA nanostructures that alter their conformation upon binding a nucleic acid analyte drastically, and reversibly, amplify the measured electrochemical signal. This amplification was controlled by the applied electrical field to achieve a response {approx} 2x104 times greater than that measured from DNA hybridization. Because the amplification is independent of the interaction between the analyte and the DNA nanostructure, our approach provides a platform for tuning the response of the system for high performance that is agnostic of the end application. These molecularly precise self-assembled DNA nanostructures when paired with scalable electronic readout can therefore lead the way to highly sensitive multiplexed biosensing.

bioengineering↗