Early-life exposures, both prenatally and in early childhood, can lead to lasting consequences in the development of health and disease throughout the life-course. Two major classes of ubiquitous chemicals detected almost universally across human populations are metals and synthetic per- and polyfluoroalkyl substances (PFAS). Metals and metalloids, such as lead and arsenic, are known hazardous substances and prenatal exposure has been shown to modulate the developing immune system. Similarly, exposure to PFAS consistently has been associated with decreased immune response to important childhood immunizations like the tetanus and diphtheria vaccines. However, our understanding of the combined impact of these two classes of chemicals on the developing immune system and immune response remains limited. While childhood immunization remains a cornerstone for public health prevention our understanding on how early-life chemical exposures affect immune system development and response remains extremely limited. Even more limited is our understanding of the joint contribution of multiple chemical exposures in early life on the immune system. Current risk assessments for these compounds are performed with adult endpoints and estimates of exposure to a single chemical. We propose to comprehensively and systematically investigate the influence of prenatal and early-life exposure to a mixture of metals and PFAS on the developing immune system and their influence on vaccine-induced immunity. We are leveraging specimens from Project Viva, a prospective birth cohort recruited in Boston, MA to 1) Determine if a prenatal mixture of first trimester metals and PFAS are individually and jointly associated as a mixture with cord blood cytokines and lymphocyte proliferation at birth; 2) Quantify the extent to which a prenatal metal/PFAS mixture is associated with Tetanus, Diphtheria and Pertussis (TDAP) and Measles, Mumps, and Rubella (MMR) vaccine titers in mid-childhood (~7 years of age) and evaluate the extent to which postnatal childhood metal and PFAS exposure contributes to this relationship; and 3) Test if DNA methylation signatures at birth measured in leukocytes mediate associations between prenatal chemical exposure and reduced TDAP and MMR vaccine antibody response in childhood. In this project we will test longitudinal associations in a large cohort of mother-child pairs with detailed confounder information and existing high-quality data and samples. We will implement novel statistical methodology to test different prenatal and postnatal environmental mixtures to comprehensively quantify the impact of chemical exposures on the developing immune system and vaccine- induced immunity. This project will provide critical knowledge on the developmental immunotoxicity of PFAS and metals, individually and as a mixture, at environmentally relevant concentrations for the U.S. population. The immune parameters of this research are clinically relevant and will help inform risk assessments for individuals as well as public health guidelines with the goal of protecting children.