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 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 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 evolution in response to vaccine pressure.
Bordetella pertussis infection has been increasing in the US, with reported cases reaching over 50,000 in 2012, a number last observed in the 1950s. Concurrently, B. pertussis lacking the pertactin protein, one of the immunogens included in the acellular vaccine formulations, has rapidly emerged since 2010, and has become the predominant circulating phenotype. Monitoring the production of the remaining acellular vaccine immunogens, such as pertussis toxin (Pt), is a critical next step. To date, methods for screening Pt have been either through genomic sequencing means or by conventional ELISAs. However, sequencing limits detection to the DNA level, missing potential disruptions in transcription or translation. Conventional ELISAs are beneficial for detecting the protein; however, they can often suffer from poor sensitivity and specificity. Here we describe a rapid, highly sensitive and specific electrochemiluminescent capture ELISA that can detect Pt production in prepared inactivated bacterial suspensions. Over 340 isolates were analyzed and analytical validation parameters, such as precision, reproducibility, and stability, were rigorously tested. Intra-plate and inter-plate variability measured at 9.8% and 11.5%, respectively. Refrigerated samples remained stable for two months and variability was unaffected (coefficient of variation was 12%). Interestingly, despite the intention of being a qualitative method, the assay was sensitive enough to detect a small, but statistically significant, difference in protein production between different pertussis promoter allelic groups of strains, ptxP1 and ptxP3. This technology has the ability to perform screening of multiple antigens at one time, thus, improving testing characteristics while minimizing costs, specimen volume, and testing time.
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