Abstract
Factor H-Binding Protein (fHbp) is an outer membrane protein antigen included in two novel meningococcal group B vaccines and, as such, is an important typing target. Approximately 50% of meningococcal disease cases in England and Wales are confirmed using real-time PCR on non-culture clinical specimens only. Protocols for typing fHbp from this subset of cases have not yet been established. Here we present a nested PCR-based assay designed to amplify and sequence fHbp from non-culture clinical specimens. From analytical sensitivity experiments carried out using diluted DNA extracts, an estimated analytical sensitivity limit of 6 fg/µL of DNA (<3 genome copies/µL) was calculated. The sensitivity of the assay was shown to be comparable to the ctrA-directed real-time PCR assay currently used to confirm invasive disease diagnoses from submitted clinical specimens. A panel of 96 diverse, patient-matched clinical specimen/isolate pairs from invasive disease cases was used to illustrate the breadth of strain coverage for the assay. All fHbp alleles sequenced from the isolates matched those derived from previous whole genome analyses. The first-round PCR primer binding sites are highly conserved, however an exceptional second-round PCR primer site mismatch in one validation isolate prevented amplification. In this case, amplification from the corresponding clinical specimen was achieved, suggesting that the use of a nested PCR procedure may compensate for any minor mismatches in round-two primer sites. The assay was successful at typing 91/96 (94.8%) of the non-culture clinical specimens in this study and exhibits sufficient sensitivity to type fHbp from the vast majority of non-culture clinical specimens received by the Meningococcal Reference Unit, Public Health England.
Highlights
Invasive meningococcal disease (IMD) represents a significant threat to the public health of communities globally
Analytical Sensitivity To assess the sensitivity of the PCR protocol to the target template, DNA was extracted from seven diverse meningococcal isolates and diluted in series to an approximate concentration of 60 ag/mL (0.025 genome copies (GC)/mL) (Table 1)
This study describes the development and validation of a sensitive PCR-based assay to aid vaccine coverage predictions and provide ongoing epidemiological surveillance data
Summary
Invasive meningococcal disease (IMD) represents a significant threat to the public health of communities globally. Vaccines containing peptide-conjugated capsular polysaccharides have been successful in reducing disease caused by capsular groups A, C, W and Y [1,2,3,4]. Disease caused by group B meningococci, remains an ever present problem and the development of vaccines against this subset has proven to be much more challenging. Formulations based upon group B outer membrane vesicles (OMVs) are yet to offer expansive immune protection due to immunodominance of the heterogeneous Porin A (PorA) antigen [6]. In a change of approach, two group B vaccines containing subcapsular, outer membrane protein antigens have recently been developed and currently represent the most promising strategy for preventing diverse group B disease [7]. Factor H-Binding Protein (fHbp) is an antigen included in both of these vaccines
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