Traditional vaccines use inactivated or live attenuated pathogens to elicit an effective adaptive immune response, but these vaccines can lack safety for immunocompromised individuals. Subunit vaccines–which display characteristic components of pathogens–are safe, stable, and readily engineered, but struggle to elicit a strong immune response. These next-generation vaccines require adjuvants (substances that stimulate the immune system) to increase efficacy. However, many currently used adjuvants lack well-understood mechanisms or wide applicability across vaccines. There is a need for new adjuvant platforms, and protein-based adjuvants are appealing because they are stable, readily engineered, and can be co-delivered with antigens on subunit vaccines. Toll-like Receptor (TLR) proteins are promising adjuvant targets that bind pathogen-associated molecules to activate the innate immune system. Of this family, TLR3 binds viral double-stranded RNA (dsRNA) and TLR5 binds the bacterial protein flagellin. Neither native agonist is a suitable adjuvant candidate; dsRNA is unstable and nonspecific and flagellin is degradation and aggregation-prone. Therefore, this project aims to design, test, and characterize novel protein-based adjuvants that can bind TLRs 3 and 5 and activate the immune system. Here, I test and characterize de novo mini-proteins that I have computationally designed to bind mouse TLR3 (mTLR3) and mouse TLR5 (mTLR5). I use yeast surface display, biolayer interferometry, and cell-surface binding assays to identify and characterize successful binders. Preliminary results show de novo mini-proteins specifically bind mTLR3 and mTLR5. Ultimately, this work hopes to provide a mouse model for these novel protein-based vaccine adjuvants with clinical aims. This project has wide-reaching public health implications, as vaccines offer the potential to improve the health and lives of countless individuals.

The emergence of the SARS-CoV-2 vaccine helped shine a light on protein subunit vaccines that use fragments of infectious protein from viruses to trigger protective immune responses. Normally in subunit vaccines, the immune system stimulation is triggered by a separately provided adjuvant. These adjuvants elicit immune responses in a broad and indeterminate manner. We hope to replace this with a molecule that can provide a more specific immune stimulation: CD40Ligand (CD40L). CD40L is an immune protein present on T cells and works to signal B cells to either replicate or create antibodies. For B cells to replicate antibodies, they need a primary signal from the antigen and a secondary signal, which CD40L initiates. To achieve this, we are using the I53-dn5 nanoparticle, which has the ability to display different ligands on its two components by putting both the antigen and adjuvant on either the pentamer or trimer component. We designed 8 different constructs where we tested two versions of CD40L, the placement of CD40L, and the linker length between CD40L and the nanoparticle surface. Out of the five stages of the project - designing, expressing, purifying, assembling, and evaluating - we have completed the first three. The designs that have expressed the best throughout each stage have been those with the CD40L truncated sequence instead of the full sequence. Furthermore, we have seen a trend with the pentamer subunit being more amenable to the addition of CD40L. Future in vitro studies and re-expressions will determine if the particle will retain stability and native CD40L function. We expect CD40L-displaying nanoparticles will promote B-cell proliferation to a greater extent than the nanoparticle vaccine displaying only hemagglutinin antigen. Ultimately, we hope to examine how co-display of CD40L with antigen will change the quality of immune response and memory in vivo.

Lacking in early emotional support has been associated with emotional dysregulation and impulsivity in adulthood. Although previous research has also demonstrated that emotion dysregulation and impulsivity are both crucial factors associated to alcohol-related problems, to our knowledge there is no study exploring the relationship between early emotional support and alcohol use in adulthood through the mechanisms of emotion dysregulation and urgency. Additionally, previous work has mostly relied on cross-sectional data. The current study aims to explore the association between early emotional support and alcohol use by assessing the role of emotion dysregulation and urgency as mediators. We use pilot data collected via cross-sectional and ecological momentary assessment (EMA) methodology. Emotion dysregulation will be assessed by the Cognitive Emotion Regulation Questionnaire (CERQ; Garnefski & Kraaij, 2001). Urgency was assessed by the averaged values of the negative and positive urgency subscales in the 59-item Impulsive Behavior Scale (UPPS-P; Whiteside & Lynam, 2001; Lynam, Smith, Whiteside, & Cyders, 2006). Early emotional support was assessed by the emotional support subscale in the Multidimensional Neglect Behavior Scale (Dubowitz et al., 2011). Alcohol use was assessed by the Daily Drinking Questionnaire (DDQ-R; Collins, Parks, & Marlatt, 1985). Longitudinal items were gathered from a subset of the above measures. For the purposes of this presentation, we plan to analyze the data using bivariate correlation analyses in preparation for a full mediation analysis in the full study. We expect to find that those lacking in emotional support during adolescence predicts both emotional dysregulation and urgency in adulthood, which leading to alcohol-related problems in adulthood. Results in the expected directions would suggest that low emotional support in adolescence may be a risk factor for substance abuse later in life, showcasing a need for caregivers to better the emotional states of their children.