Abstract: Genomic analysis reveals extensive sequence variation and hot spots of recombination in surface proteins of Streptococcus pneumoniaeWhile this phenomenon is commonly attributed to diversifying selection by host immune responses, there is little mechanistic evidence for the hypothesis that diversification of surface protein antigens produces an immune escape benefit during infection with S. pneumoniae Here, we investigate the biological significance of sequence variation within the S. pneumoniae cell wall-associated pneumococcal surface protein C (PspC) protein antigen. Using pspC allelic diversity observed in a large pneumococcal collection, we produced variant-specific protein constructs that span the sequence variability within the pspC locus. We show that antibodies raised against these PspC constructs are variant specific and prevent association between PspC and the complement pathway mediator, human factor H. We found that PspC variants differ in their capacity to bind factor H, suggesting that sequence variation within pspC reflects differences in biological function. Finally, in an antibody-dependent opsonophagocytic assay, S. pneumoniae expressing a PspC variant matching the antibody specificity was killed efficiently. In contrast, killing efficacy was not evident against S. pneumoniae expressing mismatched PspC variants. Our data suggest that antigenic variation within the PspC antigen promotes immune evasion and could confer a fitness benefit during infection.IMPORTANCE Loci encoding surface protein antigens in Streptococcus pneumoniae are highly polymorphic. It has become a truism that these polymorphisms are the outcome of selective pressure on S. pneumoniae to escape host immunity. However, there is little mechanistic evidence to support the hypothesis that diversifying protein antigens produces a benefit for the bacteria. Using the highly diverse pspC locus, we have now characterized the functional and immune implications of sequence diversity within the PspC protein. We have characterized the spectrum of biological function among diverse PspC variants and show that pspC sequence diversity reflects functional differences. Further, we show that sequence variation in PspC confers an immune escape benefit in the presence of anti-PspC variant-specific immunity. Overall, the results of our studies provide insights into the functional implications of protein sequence diversity and the role of variant-specific immunity in its maintenance.