Project Summary/Abstract Most pathogenic bacteria express surface carbohydrates called capsular polysaccharides (CPSs). CPSs are important vaccine candidates given that they are located on the outermost surface of bacteria and have distinct structures. These two features make them easily accessible and distinctly recognizable by immune surveillance, therefore resulting in the production of CPS-specific antibodies by B cells. To induce a CPS- specific adaptive immune response (i.e., T cell-mediated B cell response), CPSs are conjugated with carrier proteins, and the conjugation products are called glycoconjugate vaccines. Due to insufficient understanding of their immune activation mechanisms, current glycoconjugate vaccine strategies have reached saturation and are largely modifications of past empirical conjugation methods. The production of the current generation of glycoconjugate vaccines is based on trial and error and does not make use of specific scientific knowledge to maximize stimulation of critical immune cells (i.e., helper T cells) involved in producing protective IgG antibodies. A new perspective on carbohydrate-based vaccine research is much needed. With the potential of establishing a new paradigm, our previous discovery and preliminary data demonstrate that the mammalian CD4+ T cell repertoire contains a population of T cells that recognize carbohydrate epitopes of glycoconjugate vaccines, called Tcarbs. Building on our previous mechanistic work, we propose establishing a platform to design and develop structurally defined glycoconjugate vaccines optimized for their immunogenic and antigenic components to elicit protective immunity consistently and effectively. This proposal also addresses another key parameter controlling the immunogenicity of conjugate vaccines, which is to identify the immune correlates of protection induced by glycoconjugate vaccine immunization. In clinical practice, improving on measuring CPS-specific antibody titers and the in vitro opsonophagocytic activity as immune correlates of protection is an essential milestone to achieve for the reliable prediction of clinical efficacy. We propose to identify key helper T cell populations that are essential for vaccine efficacy as a new immune correlate of protection. Exploiting CPSs from two highly pathogenic Streptococcus pneumoniae (Spn) serotypes—Spn3 and Spn14—and building on our published and unpublished preliminary studies, we will establish a new and broadly applicable conjugate vaccine platform and characterize immune responses that result from these vaccines through two specific aims. Aim 1: Establish a knowledge-based conjugate vaccine platform composed of endolysosome-cleavable polypeptide chains chemoenzymatically conjugated with CPSs. Aim 2: Isolate and functionally characterize human Tcarb clones for their ability to induce protective humoral immunity