Screening and Genomic Characterization of Filamentous Hemagglutinin-Deficient Bordetella pertussis

Abstract

Despite high vaccine coverage, pertussis cases in the United States have increased over the last decade. Growing evidence suggests that disease resurgence results, in part, from genetic divergence of circulating strain populations away from vaccine references. The United States employs acellular vaccines exclusively, and current Bordetella pertussis isolates are predominantly deficient in at least one immunogen, pertactin (Prn). First detected in the United States retrospectively in a 1994 isolate, the rapid spread of Prn deficiency is likely vaccine driven, raising concerns about whether other acellular vaccine immunogens experience similar pressures, as further antigenic changes could potentially threaten vaccine efficacy. We developed an electrochemiluminescent antibody capture assay to monitor the production of the acellular vaccine immunogen filamentous hemagglutinin (Fha). Screening 722 U.S. surveillance isolates collected from 2010 to 2016 identified two that were both Prn and Fha deficient. Three additional Fha-deficient laboratory strains were also identified from a historic collection of 65 isolates dating back to 1935. Whole-genome sequencing of deficient isolates revealed putative, underlying genetic changes. Only four isolates harbored mutations to known genes involved in Fha production, highlighting the complexity of its regulation. The chromosomes of two Fha-deficient isolates included unexpected structural variation that did not appear to influence Fha production. Furthermore, insertion sequence disruption of fhaB was also detected in a previously identified pertussis toxin-deficient isolate that still produced normal levels of Fha. These results demonstrate the genetic potential for additional vaccine immunogen deficiency and underscore the importance of continued surveillance of circulating B. pertussis evolution in response to vaccine pressure.

Keywords: Bordetella pertussis; Fha; filamentous hemagglutinin; pertussis; whooping cough.

FIG 1

Vaccine immunogen detection by electrochemiluminescent assay. Fha (A) and Pt (B) protein production of Fha-deficient mutants J014, J199, J042, J043, and B199 compared to select Fha-producing isolates H627, I762, I959, J010, and J174 measured by secondary antibody capture. Purified protein and phosphate-buffered saline (PBS) were included for positive and negative control, respectively. Electrochemiluminescent (ECL) values are averages, and error bars represent the standard deviations for 50 replicates for Fha (A) and 18 replicates for Pt (B).

FIG 2

Fha detection by Western blotting. Fha production determined by ECL assay was found to be deficient in J014, J199, J042, J043, and B199 compared to Fha-producing isolates J010 and J365. Each well was loaded with 10 μg of total protein. The anti-Fha antibody recognized multiple polypeptides in J010, consistent with the frequent observation of various FhaB degradation products in B. pertussis cell extracts (28).

FIG 3

Observed mutations in fhaB. Mutations within domains of the FhaB preprotein: signal peptide (SP), two-partner secretion (TPS), mature C-terminal (MCD), N-terminal prodomain (PNT), proline-rich region (PRR), and extreme C-terminal (ECT) domains. Genomes of isolates J199, B199, and B3585 exhibited mutations that altered mature Fha, while J043 and J365 included IS481 insertion within the C-terminal prodomain. Predicted IS481 insertion target sites at positions 2785, 3124, and 9865 (RD16_09375 in E476) are indicated with red arrows.

FIG 4

SNP phylogeny and molecular typing of Fha-deficient isolates. Phylogenetic reconstruction of Fha-deficient isolates and select references from 584 variable nucleotides using maximum parsimony. Vaccine immunogen production, gene alleles, and PFGE profile are listed next to the tree as indicated in the key. Deficient isolates sequenced as part of the current study are highlighted in red. The scale bar indicates the number of substitutions per site.