Clustered intergenic region sequences as predictors of factor H Binding Protein expression patterns and for assessing Neisseria meningitidis strain coverage by meningococcal vaccines

Factor H binding protein (fHbp) is a major protective antigen in 4C-MenB (Bexsero®) and Trumenba®, two serogroup B meningococcal vaccines, wherein expression level is a determinant of protection. Examination of promoter-containing intergenic region (IGR) sequences indicated that nine fHbp IGR alleles covered 92% of 1,032 invasive meningococcal strains with variant 1 fHbp alleles. Relative expression values for fHbp were determined for 79 meningococcal isolates covering ten IGR alleles by quantitative reverse transcriptase polymerase chain reaction (qRT PCR). Derivation of expression clusters of IGR sequences by linear regression identified five expression clusters with five nucleotides and one insertion showing statistically associations with differences in expression level. Sequence analysis of 273 isolates examined by the Meningococcal Antigen Typing Scheme, a sandwich ELISA, found that coverage depended on the IGR expression cluster and vaccine peptide homology combination. Specific fHbp peptide-IGR expression cluster combinations were designated as 'at risk' for coverage by 4C-MenB and were detected in multiple invasive meningococcal disease cases confirmed by PCR alone and occurring in partially-vaccinated infants. We conclude that sequence-based analysis of IGR sequences is informative for assessing protein expression and has utility for culture-independent assessments of strain coverage by protein-based vaccines

Schematic representation of the <i>cbbA</i> and <i>fHbp</i> genes and intergenic regions in genomes of <i>Neisseria meningitidis</i>.

<p>The arrows indicate the transcriptional orientation of each gene. The double black arrows mark the intergenic region (IGR) containing the promoter signal of the genes and their minimum size, the double red arrow indicates the position of the <i>cbbA</i> promoter region. The nomenclature in the PubMLST Neisseria database for these sequences is as follows: <i>xerC</i>, NEIS0351; <i>cbbA</i>, NEIS0350; <i>fHbp</i>, NEIS0349; <i>cbbA</i> IGR, igr_up_NEIS0350; <i>fhbp</i> IGR, igr_up_NEIS0349 and <i>cbbA</i> promoter, pro_NEIS0350.</p

Combinatorial effects of expression level and homology to the 4C-MenB vaccine antigen for MATS-based estimates of fHbp strain coverage.

<p>All isolates (n = 132) were MenB strains with variant 1 fHbp alleles. Each symbol represents one isolate. The y-axis is the relative potency (RP) score obtained in an fHbp MATS assay. The line represents the fHbp PBT below which an isolate is defined as negative for coverage by fHbp. The x-axis is the fHbp peptide sequence and the corresponding percentage of homology observed with fHbp peptide 1.1. Expression clusters (E1 to E5) are represented by specific symbols as indicated. Samples exhibiting an RP above 0.110 are excluded from the main plot. The inset panel represents the same samples plotted with an extended y-axis range allowing inclusion of samples exhibiting RP values above 0.110.</p

Comparison of relative expression levels for <i>fHbp</i> or <i>cbbA</i> transcripts.

<p><b>Transcripts were measured by qRT PCR using <i>gdh</i> as an internal control.</b> Relative quantification (RQ) values were determined relative to strain H44/76 that was given an arbitrary value of 1. RQ values for <i>fHbp</i> (A, B, C, and G) and <i>cbbA</i> (D, E and F) were stratified by clonal complex (A, D), fHbp and cbbA peptides (B and E respectively) and IGR alleles (C, F, G).</p

fHbp expression cluster and peptide type for MenB isolates from 4C-MenB vaccine breakthrough cases.

<p>fHbp expression cluster and peptide type for MenB isolates from 4C-MenB vaccine breakthrough cases.</p