Bridging upstream and downstream for improved adenovirus 5 bioprocess.

Adenoviruses are well-known viral vectors that have been previously used in gene therapy and as a vaccine-delivery vehicle for humans and animals. During the COVID-19 pandemic, it gained renewed attention, but at the same time, it raised concerns due to side effects observed with some of the resulting vaccines administered to patients. It has been indicated that these side effects might be attributed to impurities present in the final product. Therefore, constant enhancement of the vaccine purity and further improvement of impurity detection methods are needed. In this work, we showcase an example of industry-relevant adenovirus bioprocess optimization. Our data show the effect of upstream parameters on the bioburden introduced to the downstream process. We provide an example of process optimization using a combination of the PATfix analytical method, ddPCR, infectivity, total DNA, and total protein analyses to optimize cell density, multiplicity of infection, and length of production. Additionally, we provide data illustrating the robustness of the convective interaction media quaternary amine monolithic chromatography step. This anion exchange strategy was shown to remove over 99% of protein and DNA impurities, including those unable to be addressed by tangential flow filtration, while maintaining high adenovirus recoveries.

Coiled-coil heterodimers with increased stability for cellular regulation and sensing SARS-CoV-2 spike protein-mediated cell fusion.

Coiled-coil (CC) dimer-forming peptides are attractive designable modules for mediating protein association. Highly stable CCs are desired for biological activity regulation and assay. Here, we report the design and versatile applications of orthogonal CC dimer-forming peptides with a dissociation constant in the low nanomolar range. In vitro stability and specificity was confirmed in mammalian cells by enzyme reconstitution, transcriptional activation using a combination of DNA-binding and a transcriptional activation domain, and cellular-enzyme-activity regulation based on externally-added peptides. In addition to cellular regulation, coiled-coil-mediated reporter reconstitution was used for the detection of cell fusion mediated by the interaction between the spike protein of pandemic SARS-CoV2 and the ACE2 receptor. This assay can be used to investigate the mechanism of viral spike protein-mediated fusion or screening for viral inhibitors under biosafety level 1 conditions.

A Nanoscaffolded Spike-RBD Vaccine Provides Protection against SARS-CoV-2 with Minimal Anti-Scaffold Response.

The response of the adaptive immune system is augmented by multimeric presentation of a specific antigen, resembling viral particles. Several vaccines have been designed based on natural or designed protein scaffolds, which exhibited a potent adaptive immune response to antigens; however, antibodies are also generated against the scaffold, which may impair subsequent vaccination. In order to compare polypeptide scaffolds of different size and oligomerization state with respect to their efficiency, including anti-scaffold immunity, we compared several strategies of presentation of the RBD domain of the SARS-CoV-2 spike protein, an antigen aiming to generate neutralizing antibodies. A comparison of several genetic fusions of RBD to different nanoscaffolding domains (foldon, ferritin, lumazine synthase, and ß-annulus peptide) delivered as DNA plasmids demonstrated a strongly augmented immune response, with high titers of neutralizing antibodies and a robust T-cell response in mice. Antibody titers and virus neutralization were most potently enhanced by fusion to the small ß-annulus peptide scaffold, which itself triggered a minimal response in contrast to larger scaffolds. The ß-annulus fused RBD protein increased residence in lymph nodes and triggered the most potent viral neutralization in immunization by a recombinant protein. Results of the study support the use of a nanoscaffolding platform using the ß-annulus peptide for vaccine design.

Engineering and Rewiring of a Calcium-Dependent Signaling Pathway.

An important feature of synthetic biological circuits is their response to physicochemical signals, which enables the external control of cellular processes. Calcium-dependent regulation is an attractive approach for achieving such control, as diverse stimuli induce calcium influx by activating membrane channel receptors. Most calcium-dependent gene circuits use the endogenous nuclear factor of activated T-cells (NFAT) signaling pathway. Here, we employed engineered NFAT transcription factors to induce the potent and robust activation of exogenous gene expression in HEK293T cells. Furthermore, we designed a calcium-dependent transcription factor that does not interfere with NFAT-regulated promoters and potently activates transcription in several mammalian cell types. Additionally, we demonstrate that coupling the circuit to a calcium-selective ion channel resulted in capsaicin- and temperature-controlled gene expression. This engineered calcium-dependent signaling pathway enables tightly controlled regulation of gene expression through different stimuli in mammalian cells and is versatile, adaptable, and useful for a wide range of therapeutic and diagnostic applications.