Project Summary – Project 3 (PR3) Immune development in early life (IDEAL) is dynamic and growing evidence suggests that it impacts the risk for common undesirable clinical phenotypes including low vaccine responsiveness, and proneness to respiratory infection and/or asthma. However, little is known regarding precise mechanisms nor how to redirect immune development to more favorable phenotypes. The Precision Vaccines Program (PVP) at Boston Children’s Hospital (BCH) has developed cutting-edge human in vitro assays which model age related changes in leukocyte function- i.e., immune ontogeny and population (e.g. age)-specific effects and mechanisms of action of immunomodulatory agents, including metabolites, proteins, adjuvants and vaccines. Project 3 (PR3) will leverage human in vitro modeling to gain insight into signaling pathways that are relevant to the clinical phenotypes observed. Our published and unpublished preliminary data indicate that our sample-sparing human in vitro assay platforms can model innate and adaptive immune responses of infants and young children which vary by age and disease status. Our hypothesis is that our innate and adaptive in vitro modeling platforms can meaningfully interrogate molecular signaling pathways relevant to endotypes (disease sub-types) of clinical phenotypes such as vaccine responsiveness, respiratory infection and/or asthma. Our goal is to leverage our cutting-edge human in vitro assay systems to model human immune cell responses to infant vaccines as well as to confirm, assess and translate the pathways identified in PR1 and PR 2. We will achieve this goal by pursuing the following Specific Aims (SAs): SA1. Assess mechanisms underlying IDEAL endotypes. In this aim we will pursue molecular interrogation of pathways, biomarkers, metabolites discovered in PR1 and PR2. SA2. Identify immunomodulators that re-direct trajectories from unfavorable to favorable endotypes. We will model immune activation in response to agents capable of re- shaping immune endotypes, including agents that impact the relevant endotype-associated pathways identified in PR1 and PR2 . Overall, successful completion of PR3 will provide fresh insight into IDEAL in relation to vaccine responsiveness, infection and/or asthma proneness. This effort will provide mechanistic insight into IDEAL, help confirm and probe novel prognostic biomarkers and pathways identified in PR1 and PR2, and identify agents (e.g., proteins, metabolites, adjuvants, vaccines) that can redirect human infant leukocytes away from unfavorable endotypes associated with low vaccine responsiveness, respiratory infection and/or asthma and towards favorable trajectories and endotypes thereby advancing child health.