Production and Purification of Filovirus Glycoproteins.

Ebola (EBOV) and Marburg (MARV) viruses cause hemorrhagic fever disease in humans and non-human primates (NHPs) with case-fatality rates as high as 90%. The 2013-2016 Ebola virus disease (EVD) outbreak led to over 28,000 cases and 11,000 deaths and took an enormous toll on the economy of West African nations, in the absence of any vaccine or therapeutic options. Like EVD, there have been at least 6 outbreaks of MVD with ~88% case-fatality and the most recent cases emerging in Equatorial Guinea in February 2023. These outbreaks have spurred an unprecedented global effort to develop vaccines and therapeutics for EVD and MVD and led to an approved vaccine (ERVEBO™) and two monoclonal antibody (mAb) therapeutics for EBOV. In contrast to EVD, therapeutic options against Marburg and another Ebola-relative Sudan virus (SUDV) are lacking. The filovirus glycoprotein (GP), which mediates host cell entry and fusion, is the primary target of neutralizing antibodies. In addition to its pre- and post-fusion trimeric states, the protein is highly glycosylated making production of pure and homogeneous trimers on a large scale, a requirement for subunit vaccine development, a challenge. In efforts to address this roadblock, we have developed a unique combination of structure-based design, selection of expression system, and purification methods to produce uniform and stable EBOV and MARV GP trimers at scales appropriate for vaccine production.

Antigenic landscapes on Staphylococcus aureus pore-forming toxins reveal insights into specificity and cross-neutralization.

Staphylococcus aureus carries an exceptional repertoire of virulence factors that aid in immune evasion. Previous single-target approaches for S. aureus-specific vaccines and monoclonal antibodies (mAbs) have failed in clinical trials due to the multitude of virulence factors released during infection. Emergence of antibiotic-resistant strains demands a multi-target approach involving neutralization of different, non-overlapping pathogenic factors. Of the several pore-forming toxins that contribute to S. aureus pathogenesis, efforts have largely focused on mAbs that neutralize α-hemolysin (Hla) and target the receptor-binding site. Here, we isolated two anti-Hla and three anti-Panton-Valentine Leukocidin (LukSF-PV) mAbs, and used a combination of hydrogen deuterium exchange mass spectrometry (HDX-MS) and alanine scanning mutagenesis to delineate and validate the toxins' epitope landscape. Our studies identified two novel, neutralizing epitopes targeted by 2B6 and CAN6 on Hla that provided protection from hemolytic activity in vitro and showed synergy in rodent pneumonia model against lethal challenge. Of the anti-LukF mAbs, SA02 and SA131 showed specific neutralization activity to LukSF-PV while SA185 showed cross-neutralization activity to LukSF-PV, γ-hemolysin HlgAB, and leukotoxin ED. We further compared these antigen-specific mAbs to two broadly neutralizing mAbs, H5 (targets Hla, LukSF-PV, HlgAB, HlgCB, and LukED) and SA185 (targeting LukSF-PV, HlgAB, and LukED), and identified molecular level markers for broad-spectrum reactivity among the pore-forming toxins by HDX-MS. To further underscore the need to target the cross-reactive epitopes on leukocidins for the development of broad-spectrum therapies, we annotated Hla sequences isolated from patients in multiple countries for genomic variations within the perspective of our defined epitopes.

Rapid exploration of the epitope coverage produced by an Ebola survivor to guide the discovery of therapeutic antibody cocktails.

BACKGROUND: Development of successful neutralizing antibodies is dependent upon broad epitope coverage to increase the likelihood of achieving therapeutic function. Recent advances in synthetic biology have allowed us to conduct an epitope binning study on a large panel of antibodies identified to bind to Ebola virus glycoprotein with only published sequences. METHODS AND RESULTS: A rapid, first-pass epitope binning experiment revealed seven distinct epitope families that overlapped with known structural epitopes from the literature. A focused set of antibodies was selected from representative clones per bin to guide a second-pass binning that revealed previously unassigned epitopes, confirmed epitopes known to be associated with neutralizing antibodies, and demonstrated asymmetric blocking of EBOV GP from allosteric effectors reported from literature. CONCLUSIONS: Critically, this workflow allows us to probe the epitope landscape of EBOV GP without any prior structural knowledge of the antigen or structural benchmark clones. Incorporating epitope binning on hundreds of antibodies during early stage antibody characterization ensures access to a library's full epitope coverage, aids in the identification of high quality reagents within the library that recapitulate this diversity for use in other studies, and ultimately enables the rational development of therapeutic cocktails that take advantage of multiple mechanisms of action such as cooperative synergistic effects to enhance neutralization function and minimize the risk of mutagenic escape. The use of high-throughput epitope binning during new outbreaks such as the current COVID-19 pandemic is particularly useful in accelerating timelines due to the large amount of information gained in a single experiment.

HER3-Targeted Affibodies with Optimized Formats Reduce Ovarian Cancer Progression in a Mouse Xenograft Model.

Expression of the receptor tyrosine kinase HER3 is negatively correlated with survival in ovarian cancer, and HER3 overexpression is associated with cancer progression and therapeutic resistance. Thus, improvements in HER3-targeted therapy could lead to significant clinical impact for ovarian cancer patients. Previous work from our group established multivalency as a potential strategy to improve the therapeutic efficacy of HER3-targeted ligands, including affibodies. Others have established HER3 affibodies as viable and potentially superior alternatives to monoclonal antibodies for cancer therapy. Here, bivalent HER3 affibodies were engineered for optimized production, specificity, and function as evaluated in an ovarian cancer xenograft model. Enhanced inhibition of HER3-mediated signaling and increased HER3 downregulation associated with multivalency could be achieved with a simplified construct, potentially increasing translational potential. Additionally, functional effects of affibodies due to multivalency were found to be specific to HER3 targeting, suggesting a unique molecular mechanism. Further, HER3 affibodies demonstrated efficacy in ovarian cancer xenograft mouse models, both as single agents and in combination with carboplatin. Overall, these results reinforce the potential of HER3-targeted affibodies for cancer therapy and establish treatment of ovarian cancer as an application where multivalent HER3 ligands may be useful. Further, this work introduces the potential of HER3 affibodies to be utilized as part of clinically relevant combination therapies (e.g., with carboplatin).

Protein-based vehicles for biomimetic RNAi delivery.

Broad translational success of RNA interference (RNAi) technology depends on the development of effective delivery approaches. To that end, researchers have developed a variety of strategies, including chemical modification of RNA, viral and non-viral transfection approaches, and incorporation with delivery vehicles such as polymer- and lipid-based nanoparticles, engineered and native proteins, extracellular vesicles (EVs), and others. Among these, EVs and protein-based vehicles stand out as biomimetically-inspired approaches, as both proteins (e.g. Apolipoprotein A-1, Argonaute 2, and Arc) and EVs mediate intercellular RNA transfer physiologically. Proteins specifically offer significant therapeutic potential due to their biophysical and biochemical properties as well as their ability to facilitate and tolerate manipulation; these characteristics have made proteins highly successful translational therapeutic molecules in the last two decades. This review covers engineered protein vehicles for RNAi delivery along with what is currently known about naturally-occurring extracellular RNA carriers towards uncovering design rules that will inform future engineering of protein-based vehicles.