{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,1,1]],"date-time":"2026-01-01T13:42:41Z","timestamp":1767274961366,"version":"3.48.0"},"posted":{"date-parts":[[2025,12,17]]},"group-title":"Microbiology","reference-count":73,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2025,12,17]],"date-time":"2025-12-17T00:00:00Z","timestamp":1765929600000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2025,12,17]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  <jats:italic>Streptococcus pneumoniae<\/jats:italic>\n                  (Spn) colonization of the nasopharynx is a prerequisite for transmission and invasive disease. To investigate how repeated asymptomatic colonization shapes immunity and influences bacterial traits, we developed the Repeated Asymptomatic Murine Pneumococcal Colonization (RAMPC\u2083) model using strains belonging to serotypes: 2 (D39), 3 (WU2), and 4 (TIGR4). Sequential colonization revealed strain- and exposure-order\u2013dependent effects on bacterial burden, with initial colonization yielding robust carriage and subsequent exposures resulting in diminished burden and rapid clearance. Humoral profiling demonstrated antigenic imprinting: the first colonizing strain largely determined IgG and IgA specificity, with minimal diversification after repeated exposures. Reactivity was strongest for biofilm-associated antigens correlating with each strain\u2019s biofilm-forming capacity. Using TIGR4 mutants deficient in biofilm formation, we confirmed that in vivo aggregate formation drives humoral responses. Human sera from naturally colonized adults mirrored these findings, favoring biofilm antigens independent from capsule. Protection was demonstrated as triple-colonized mice exhibited reduced mortality and bacteremia following pneumococcal pneumonia challenge. Moreover, the initial colonizing strain influenced protection against heterologous infection, underscoring the lasting imprint of the biofilm phenotype on immunity. Finally, IgA responses in nasal-associated lymphoid tissue paralleled serum IgA patterns, validating systemic measurements as a proxy for mucosal immunity. Collectively, these results reveal that biofilm formation during colonization is a key determinant of humoral immunity and protection, providing insight into pneumococcal biology and informing strategies to design next-generation interventions.\n                <\/jats:p>\n                <jats:sec>\n                  <jats:title>Author summary<\/jats:title>\n                  <jats:p>\n                    <jats:italic>Streptococcus pneumoniae<\/jats:italic>\n                    (Spn) is a leading cause of pneumonia, meningitis, and sepsis, yet its primary lifestyle is asymptomatic colonization of the nasopharynx. Understanding how colonization shapes immunity and bacterial physiology is critical for predicting disease risk and improving future vaccines. Using a novel murine model of repeated colonization and human sera from naturally colonized adults, we show that humoral immunity is strongly biased toward antigens expressed during biofilm growth, the predominant mode of\n                    <jats:italic>Spn<\/jats:italic>\n                    in the nasopharynx, rather than planktonic forms. This response is strain-dependent and imprinted by the first colonizing strain, influencing subsequent exposures and protection against pneumonia. Importantly, biofilm formation, not capsule, drives immune recognition revealing a key link between bacterial physiology and host immunity. These findings provide fundamental insight into pneumococcal biology and the host response, suggesting that targeting biofilm-associated antigens may improve vaccine design or strategies to prevent transmission and invasive disease.\n                  <\/jats:p>\n                <\/jats:sec>","DOI":"10.64898\/2025.12.17.694863","type":"posted-content","created":{"date-parts":[[2025,12,18]],"date-time":"2025-12-18T15:06:21Z","timestamp":1766070381000},"source":"Crossref","is-referenced-by-count":0,"title":["Biofilm formation during pneumococcal carriage imprints naturally acquired humoral immunity"],"prefix":"10.64898","author":[{"given":"Jessica R.","family":"Lane","sequence":"first","affiliation":[{"name":"Department of Microbiology, Heersink School of Medicine, The University of Alabama at Birmingham, Birmingham, Alabama, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Henry","family":"Mauser","sequence":"additional","affiliation":[{"name":"Department of 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