Abstract Description Nasopharyngeal colonization of Streptococcus pneumoniae (Spn) is a prerequisite for infection and an important step in developing pneumococcal immunity. Studies report that co-infection with pneumococcus and influenza vaccine can increase myeloid cell responsiveness to pneumococcus and other respiratory bacterial pathogens but decrease vaccination efficacy. To study mechanisms of potential synergy, we used live attenuated influenza (LAIV) to safely simulate influenza infection in the human nose, followed by an experimental human pneumococcal challenge (EHPC). 30-90 days post the nasal challenge, bronchoalveolar lavage samples were collected. By analyzing single-cell RNA-seq of over 80,000 myeloid cells from volunteers who received LAIV (n = 10), inactivated influenza vaccine (control, n = 11), or no-vaccine (n = 4), we have identified important macrophage populations and their molecular signatures that may play a major role on protection against pneumonia with or without viral challenge. We hope this immunological knowledge could improve vaccine efficacy by disrupting viral–bacterial synergy. Funding Sources Gates Foundation Topic Categories Microbial, Parasitic, and Fungal Immunology (MPF)