CC180 clade dynamics does not universally explain Streptococcus pneumoniae serotype 3 persistence post-vaccine: a global comparative population genomics study

BackgroundClonal complex 180 (CC180) is currently the major clone of serotype 3 Streptococcus pneumoniae (Spn). The 13-valent pneumococcal conjugate vaccine (PCV13) does not have significant efficacy against serotype 3 despite polysaccharide inclusion in the vaccine. It was hypothesized that PCV13 may effectively control Clade I of CC180 but that Clades III and IV are resistant, provoking a population shift that enables serotype 3 persistence. This has been observed in the United States, England, and Wales but not Spain. We tested this hypothesis further utilizing a dataset from Portugal. MethodsWe whole-genome sequenced (WGS) 501 serotype 3 strains from Portugal isolated from patients with pneumococcal infections between 1999-2020. The draft genomes underwent phylogenetic analyses, pangenome profiling, and a genome-wide association study (GWAS). We also completed antibiotic susceptibility testing and compiled over 2,600 serotype 3 multilocus sequence type 180 (MLST180) WGSs to perform global comparative genomics. FindingsCC180 Clades I, II, III, IV, and VI distributions were similar when comparing non-invasive pneumonia isolates and invasive disease isolates (Fishers exact test, P=0.29), and adult and pediatric cases (Fishers exact test, P=0.074). The serotype 3 CCs shifted post-PCV13 (Fishers exact test, P<0.0001) and Clade I became dominant. Clade I is largely antibiotic-sensitive and carries the {Phi}OXC141 prophage but the pangenome is heterogenous. Strains from Portugal and Spain, where Clade I remains dominant post-PCV13, have larger pangenomes and are associated with the presence of two genes encoding hypothetical proteins. InterpretationClade I became dominant in Portugal post-PCV13, despite the burden of the prophage and antibiotic sensitivity. The accessory genome content may mitigate these fitness costs. Regional differences in Clade I prevalence and pangenome heterogeneity suggest that clade dynamics is not a generalizable approach to understanding serotype 3 vaccine escape. FundingNational Institute of Child Health and Human Development, Pfizer, and Merck Sharp & Dohme. Research in contextO_ST_ABSEvidence before this studyC_ST_ABSWe conducted this study because of the mounting interest surrounding the changing prevalence of serotype 3 Streptococcus pneumoniae (Spn) genetic lineages and the potential association with escape from 13-valent pneumococcal conjugate vaccine (PCV13) control. To inform our investigation, we searched the PubMed database using different combinations of the following keywords: "Streptococcus pneumoniae", "serotype 3", "CC180", "PCV13", "Clade I", "Clade I{beta}", and "Clade II". The search included all English language primary research articles published before July 1st, 2024; this language limitation may bias the results of our assessment. Most ST3 isolates belong to clonal complex 180 (CC180), and one study identified three major lineages within CC180: Clade I, Clade I{beta}, and Clade II. This study observed a global trend of increasing Clade II prevalence with a concomitant decrease in Clade I prevalence over time, which was associated with the introduction of PCV13 in the United States. A report from England and Wales made a similar observation. It was therefore hypothesized that PCV13 may be effective at controlling Clade I and that Clade II is driving vaccine escape. Later work refined the clade classification system as follows: Clade I (Clade I), Clades II and VI (Clade I{beta}), Clades III and IV (Clade II), and Clade V. Clade I strains are marked by a significantly lower recombination rate partly due to the presence of a lineage-specific prophage interfering with competence development, which is a potential mechanism explaining the possible reduced fitness of Clade I. Clade I is also noted to be mostly antibiotic-susceptible. However, a recent study found that Clade I persists as a dominant serotype 3 lineage in Spain, so the generalizability and implications of clade dynamics remain unclear. Added value of this studyEarly work assessing the association between changes in serotype 3 clade prevalence and PCV13 was limited by small sample sizes. In addition, studies investigating differences in clade dynamics did not comprehensively consider patient age or disease manifestations such as non-invasive pneumonia and invasive infections. In this study, we evaluated 501 serotype 3 strains from Portugal to investigate clade dynamics. This must be explored in different geographic contexts for a more robust understanding of changing serotype 3 population genomics. We also sought to define genetic determinants linked to strains from regions in which Clade I remains dominant. This is an important step towards a more mechanistic understanding of the serotype 3 CC180 lineage fitness landscape. Implications of all the available evidenceUnlike other serotypes covered by PCV13, serotype 3 has evaded vaccine control. It has been suggested that Clade I prevalence has decreased due to PCV13, which has created an expanded niche for strains from other clades and ultimately renders PCV13 less effective against serotype 3. This postulation has important implications for the future design of an improved vaccine, so this hypothesis must be thoroughly tested in diverse contexts. We find that Clade I remains the dominant lineage in Portugal even after the introduction of PCV13. We delineate Clade I pangenome heterogeneity and show that strains from Portugal and Spain share similar pangenome features in contrast to Clade I strains from regions where Clade I decreased in prevalence, which should motivate future studies to elucidate more generalizable population genomics trends that may better inform strategies for the design of an improved vaccine.