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Streptoccocus pneumoniae is a leading cause of pneumonia, sepsis and meningitis worldwide and kills over 300,000 children under 5 globally per year [1]. Glycoconjugate vaccines, such as Prevnar 13 and Prevnar 20, have proven safe and effective at preventing infection from the most prevalent serotypes of S. pneumoniae. Composed of a pathogen-specific glycan antigen attached to an immunogenic carrier protein, conjugate vaccines train the immune system to recognize the glycans that are natively displayed on the outside of bacteria. Current methods, however, for producing conjugate vaccines require expensive equipment, growing the pathogenic bacteria of interest, and cold-chain distribution. Cell-free systems circumvent many challenges posed by centralized manufacturing by utilizing crude extracts derived from bacteria to produce any protein of interest via DNA template. Recent work has developed cell-free glycoprotein synthesis (CFGpS) which modifies CFPS for producing glycoconjugate vaccines. In CFGpS, a lysate enriched with both the glycan of interest and the oligosaccharyltransferase (OST), which enzymatically transfers glycans onto proteins, is used to produce the carrier protein of interest [2]. In this work, we modify the CFGpS platform to produce a candidate conjugate vaccine against S. pneumoniae serotype 4. We began by improving glycosylation efficiency by using a mutated PglB (OST) to transfer CPS 4. We then further improve glycosylation efficiency by using extract derived from a glycoengineered strain of E. coli for increased production of the CPS 4 glycan [5]. Lastly, we demonstrate that by incorporating these two changes, we can efficiently transfer the S. pneumoniae CPS 4 glycan onto an FDA approved carrier protein, Protein D. Future work will focus on expanding the CFGpS platform to other serotypes of S. pneumoniae to enable production of a protective, multivalent pneumococcal vaccine. Overall, these promising results open the door to increased access to vaccines against S. pneumoniae in low resource settings.