Objectives/Goals: Develop a cervicovaginal mucus replacement to prevent bacterial vaginosis (BV) in pregnant women. Our therapeutic, specialized hydrogel in natural enhancement for vaginal health (SHINE-VH), is formulated through polymer chemistry, tested for efficacy and safety through microbiology, and translated through clinical and translational science. Methods/Study Population: We will develop SHINE-VH with optimized viscoelastic and mucoadhesive properties intended to mimic healthy vaginal mucus. SHINE-VH will be synthesized via robust photoiniferter methods and investigated through shear rheology, sugar binding, and permeability studies. To evaluate the biocompatibility and safety profile of SHINE-VH, we will utilize a series of in vitro models to test its impact on the viability and cytotoxicity of human vaginal epithelial cells. In addition, we will assess the capacity of SHINE-VH to fortify vaginal barrier integrity and modulate anti-inflammatory activities in a 2D epithelial barrier model exposed to BV-associated pathogens. Lastly, employing organ-on-a-chip technology, vaginal swabs from both healthy and suspected BV pregnant patients will be treated with SHINE-VH. Results/Anticipated Results: SHINE-VH will mimic the protective, hydrophilic gel network of natural mucins. The viscoelastic properties of our formulation determined by shear rheology will be tuned through concentration and polymer composition to mimic vaginal mucus. We will also show the facile movement of small molecule nutrients through the SHINE-VH network via sugar-binding and permeability tests. Additionally, we anticipate that the introduction of SHINE-VH, due to their xenobiotic nature as synthetic mucins, will modulate the microbiota by diminishing inflammation, thereby reinforcing the cervicovaginal mucus and cultivating a vaginal microbiome that is more resilient to the disruptive impacts of BV. Such modulation could lead to a marked difference between the SHINE-VH-treated and untreated groups. Discussion/Significance of Impact: BV affects ~30% of women globally and is associated with severe gynecologic and obstetric complications, representing a significant unmet need in women’s health. SHINE-VH offers a novel approach to BV management, aiming to strengthen vaginal mucosal integrity, potentially reducing BV prevalence, and improving women’s health outcomes.