DNA nanotechnology is ideally suited for numerous applications from the crystallization and solution of macromolecular structures to the targeted delivery of therapeutic molecules. The foundational goal of structural DNA nanotechnology was the development of a lattice to host proteins for crystal structure solution. To further progress towards this goal, 36 unique four-armed DNA junctions were designed and crystallized for eventual solution of their 3D structures. While most of these junctions produced macroscale crystals which diffracted successfully, several prevented crystallization. Previous results used a fixed isomer and subsequent investigations adopted an alternate isomer to investigate the impact of these small sequence changes on the stability and structural properties of these crystals. DNA nanotechnology has also shown promise for a variety biomedical applications. In particular, DNA origami has been demonstrated as a promising tool for targeted and efficient delivery of drugs and vaccines due to their programmability and addressability to suit a variety of therapeutic cargo and biological functions. To this end, a previously designed DNA barrel nanostructure with a unique multimerizable pegboard architecture has been constructed and characterized via TEM for later evaluation of its stability under biological conditions for use in the targeted delivery of cargo, including CRISPR-containing adeno-associated viruses (AAVs) and mRNA.

In this study, we investigated the inactivation of wild-type vMyx-GFP (MYXV) using different methods. Assays were performed in vitro to test the following inactivation methods: heat, longwave UV only, longwave UV with psoralen (P + LWUV), and psoralen (P) only. In vitro assays demonstrated that the psoralen alone treatment did not cause any inactivation. These results showed that effective inactivation using psoralen was likely reliant on subsequent UV irradiation, creating a synergistic effect. Additionally, the UV and P + LWUV treatment demonstrated inactivation of MYXV, although by different mechanisms, as the UV-only treated virus demonstrated background infection, while P + LWUV treated virus did not. In mice, P + LWUV and UV treatment of MYXV demonstrated to be effective inactivation methods and likely preserved the antigenic epitopes of MYXV, allowing for the production of neutralizing antibodies in mice. More research is recommended on the heat treatment of MYXV as neutralizing antibodies were not observed, possibly due to the treatment denaturing antigenic epitopes or needing more booster injections to reach the threshold antibody concentration for protection. Furthermore, we demonstrated that the intraperitoneal (IP) injection of inactivated MYXV was superior to the subcutaneous injection in eliciting a strong immune response. The increased neutralizing antibodies observed after IP injection could be due to the advantage that the IP route has of reaching lymphoid tissue faster.

Memory CD8+ T cells protect against secondary viral infections. They develop and maintain exclusively in circulation (e.g. central memory - Tcm) or are excluded from re-circulation (resident memory - Trm). The extracellular ATP receptor P2RX7 promotes both Tcm and Trm generation. High (P2RX7hi) P2RX7-expressing early effector cells show survival, memory and pluripotency genes. Conversely, many terminal effector (TE) and apoptosis genes are upregulated in low (P2RX7lo) P2RX7-expressing cells. Among these genes is the zinc-finger transcriptional repressor Zeb2, which promotes TE differentiation at the expense of the memory cell pool. Given that Zeb2 was higher in P2RX7lo early effector cells, we postulated that Zeb2 ablation would allow P2RX7-deficient CD8+ T cells to skew towards memory subsets. To test this, we used RNP-based CRISPR-Cas9 to knockout Zeb2 in wild type or P2RX7-deficient P14 cells. At the memory timepoint, Zeb2 ablation led to a rescue of the ability of P2RX7-deficient cells to differentiate into the CD62L+ Tcm and CD69hiCD103hi Trm subsets, as well as increase the population of each. Our data suggest that P2RX7 imprints a pro-memory signature that is, to some extent, dependent on the negative regulation of Zeb2.

Plant-made virus-like particles (VLPs), composed of HIV-1 Gag and deconstructed gp41 proteins, have been shown to be safe and immunogenic in mice. Here, we report the successful production of HIV-1 Gag/dgp41 VLPs in Nicotiana benthamiana, using an enhanced geminivirus-based expression vector. This novel vector results in unique expression kinetics, with peak protein accumulation and minimal necrosis achieved on day 4 post-infiltration. In comparing various purification strategies, it was determined that a 20% ammonium sulfate precipitation is an effective and efficient method for removing plant proteins and purifying the recombinant VLPs of interest. If further purification is required, this may be achieved through ultracentrifugation. VLPs are a useful platform for a variety of biomedical applications and developing the technology to efficiently produce VLPs in the plant expression system is of critical importance.

The COVID 19 pandemic has highlighted the necessity of accurately and simply relying scientific discovery and information to the public. Among scientists, the practice is to reduce jargon, engage the audience through storytelling, and include enough detail to give a broad understanding of a narrow topic. Conflict between journalists and scientists leads to a creation of a different narrative for the general public. The news site CNN.com was searched with the google archive function by year for articles that included the keyword vaccine. Articles were sorted into categories of main focus such as political, cultural and scientific or mixed. Results were analyzed and conclusions made about the amount of content in each category for the kind of narrative being written about vaccines, with most years having most articles in the political category. Possible effectiveness of mixed categories were discussed and areas future research identified.