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.

Group B Streptococci (GBS) are gram-positive bacteria that asymptomatically colonizes the vaginal tract of approximately 18% of women worldwide. However, during pregnancy GBS in the lower genital tract can ascend into the uterus and infect the placenta and baby resulting in preterm birth, stillbirth, and neonatal infection. We have used a nonhuman primate (NHP; pigtail macaque, Macaca nemestrina) model to determine differences between GBS strains that confer different levels of invasiveness. The objective of the study was to determine if there were differences in gene expression among animals infected with a “progressive” versus a “localized/resolved” infection. We hypothesized that a greater inflammatory response would be associated with a “progressive” GBS infection compared to the “localized/resolved”. Twenty one NHP received either a choriodecidual inoculation of: 1) 1-3 X 10^8 colony forming units (CFU) of hypervirulent GBSΔcovR (n=15) or, 2) saline (n=6). Cesarean section was performed at preterm labor or 1-3 days after GBS infection or 7 days after saline inoculation. Placental chorioamniotic membranes were sampled near the inoculation site. GBS infections were categorized as “progressive infections”, “localized/resolved infections”, or “resolved” infections at the time of preterm labor or 3 days after GBS inoculation. Next, we prepared mRNA libraries from placental chorioamniotic membranes near the GBS inoculation site, which were sequenced using the NextSeq 550 platform. Data were normalized and then analyzed by Single Gene Analysis, Gene Set Analysis, and Ingenuity Pathway Analysis. The analysis is currently ongoing and will be ready to summarize during the “Revision Window”. Prevention of GBS infection in pregnancy is complex and is likely influenced by multiple factors, including pathogenicity, host factors, and the vaginal microbiome. Understanding mechanisms influencing the invasiveness of GBS infections during pregnancy will facilitate the development of novel therapeutics and vaccines.