PROJECT SUMMARY/ABSTRACT Vaccination against infectious disease saves an estimated 4 million lives each year and is one of the most effective and cost-effective medical advances in human history. Nevertheless, infectious disease remains a leading cause of mortality worldwide due to (1) the lack of vaccines formulations that confer long-lived immunity and (2) vaccine accessibility challenges. Adjuvants are capable of enhancing the immune response to antigens; however, there are limits to their efficacy. For example, alum, the most common clinical adjuvant, is known to enhance humoral immunity but have relatively little effect on cellular immunity. In addition to exploring new adjuvants, recent studies have begun exploring the impact of antigen release kinetics on the immune response. Using infusion pumps or sequential injections to administer antigen over days to weeks, researchers have demonstrated that exponentially increasing antigen release kinetics over a period of days to weeks can significantly enhance the immune response compared to one bolus injection despite using the same cumulative antigen dose. These kinetics better mimic a native infection, in which the pathogen replicates, exposing immune cells to increasing quantities of antigen over time until the infection is cleared, thereby aligning with the kinetics our immune system has evolved to respond to. Unfortunately, replacing each injection of soluble antigen currently administered in the clinic with a series of injections spread over days or the continuous use of an infusion pump for days to weeks is not clinically viable due to cost, invasiveness, and accessibility issues. In this proposal, we will develop vaccine formulations that exhibit exponential release to mimic the antigen kinetics of native infections, thereby enhancing vaccine potency without alterations in clinical practice. We have developed a unique fabrication method, termed Particles Uniformly Liquified and Sealed to Encapsulate Drug (PULSED), that produces biodegradable microparticles that exhibit pulsatile release after a material-dependent delay. Importantly, this system is fully modular, so we can combine different particle populations to construct the desired release profile, overcoming the constraints of other passive vaccine delivery systems whose rates of antigen release inherently decrease over time. We will first identify microparticle compositions that release diphtheria toxoid (DT) at discrete time points within two weeks in vitro and in vivo. Next, we will optimize microparticle processing conditions and incorporate stabilizing excipients to ensure DT is released in its immunity-conferring conformation. Lastly, we will evaluate the humoral and cellular immune responses to combinations of PULSED particles that, together, exhibit exponential antigen release and compare those responses to responses elicited by a soluble dose of vaccine as well as microparticle populations constructed to achieve other antigen ...