Distribution of NHBA peptides in the 300 isolates panel.

<p>Others^a: NHBA peptides not present in more than one isolate (peptides 8, 9, 13, 25, 47, 53, 115, 160, 187, 237, 304, 307, 308, 309, 310, 355, 367, 368, 370, 385, 460, 461, 462, 463, 464, 465, 468, 469, 470, 471). Others cc^b: Others clonal complexes. NA^c: Clonal complexes non assigned.</p

Distribution of clonal complexes and main PorA genotypes in the 300 isolates panel.

<p>*NA: Clonal complex non assigned.</p

Distribution of MATS strains predicted to be covered by cc and specific vaccine antigen combinations.

<p>Strains were defined as covered if they possessed PorA P1.4 or had relative potency greater than the positive bactericidal threshold for fHbp, NHBA or NadA. *NA: Clonal complex non assigned.</p

Distribution of fHbp variant families and peptides in the 300 isolates panel.

<p>Others¹: FHbp variant family 1 peptides not present in more than three isolates (peptides 4, 15, 35, 37, 54, 65, 90, 87, 108, 110, 144, 213, 218, 236, 252,275, 322, 357, 358, 359, 360, 361, 362, 363, 365, 373, 374, 403, 456, 480, 544, 545). Others²: FHbp variant family 2 peptides not present in more than three isolates (peptides 18, 24, 34, 104, 367, 548, 551). Others³: FHbp variant family 3 peptides not present in more than three isolates (peptides 29, 31, 174, 188, 294, 364, 366, 368, 398, 399, 400,401,402, 485, 494, 532, 536, 549, 550, 552, 555). Others cc*: Others clonal complexes, included clonal complexes non assigned.</p

Distribution of NadA variants and peptides in the 300 isolates panel.

<p>Frameshift¹: The <i>nadA</i> nucleotide allele presents a frameshift mutation resulting in a premature stop codon (variant 1 allele 44; variant 4/5 alleles 12, 34, 38, 39, 40 and 46; variant 6 alleles 37, 43 and 94). IS1301²: The <i>nadA</i> gene (allele 50) is disrupted by an insertion sequence IS1301. NA³: Clonal complex non assigned.</p

An Analysis of the Sequence Variability of Meningococcal fHbp, NadA and NHBA over a 50-Year Period in the Netherlands

<div><p>Studies of meningococcal evolution and genetic population structure, including the long-term stability of non-random associations between variants of surface proteins, are essential for vaccine development. We analyzed the sequence variability of factor H-binding protein (fHbp), Neisserial Heparin-Binding Antigen (NHBA) and <i>Neisseria</i> adhesin A (NadA), three major antigens in the multicomponent meningococcal serogroup B vaccine 4CMenB. A panel of invasive isolates collected in the Netherlands over a period of 50 years was used. To our knowledge, this strain collection covers the longest time period of any collection available worldwide. Long-term persistence of several antigen sub/variants and of non-overlapping antigen sub/variant combinations was observed. Our data suggest that certain antigen sub/variants including those used in 4CMenB are conserved over time and promoted by selection.</p></div

Associations between different loci.

<p>The Cramer’s V coefficient was used to measure association statistics with clonal complexes. The values indicated a strong association between antigen alleles and clonal complexes. The Standardized Index of Association <i>I_A^S</i> was used to test the stability of associations between different loci. The antigen pairs showed relatively high <i>I_A^S</i> values, indicative of strong, stable associations between the different loci over time. Moreover, the combinations showed a non-overlapping structure (measured by f* metrics) that suggested the immune selection maintaining antigenic combinations.</p>§<p>The three statistical parameters V, <i>I_A^S</i> and <i>f*</i> are based on the frequency of the alleles and vary between 0 (random distribution) and 1 (perfect association or non overlapping distribution in the case of <i>f*</i>).</p

The evolution of cc41/44 over 50 years.

<p>The structure of cc41/44 was analysed with phyloviz based on the goeBURST algorithm <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065043#pone.0065043-Francisco1" target="_blank">[62]</a>. Each number correspond to a different ST, the sizes of circles are proportional to the number of isolates. <b>A) fHbp variants.</b> In 1960-1970, when the ST-44 sub-complex was predominant, fHbp-2 and fHbp-3 were most represented. After 1980, when the ST-41 sub-complex was prevalent, fHbp-1 became the most frequent. Of note, independently from its prevalence, fHbp-1 was the only variant that was present in both sub-complexes at all time periods. <b>B) NHBA</b>. NHBA-2, the most frequent in cc41/44 (82%), was equally shared by the two sub-complexes from 1960 to 2008-2009.</p

Persistence of the fHbp, NHBA and NadA sub/variants included in 4CMenB.

<p>fHbp-1.1 and NadA-3.8 persisted for thirty years, NHBA-2 for fifty years.</p

Longevity of the most frequent combinations of two and three antigen sub/variants.

<p>Out of the 9 most frequent combinations observed, several persisted for at least twenty years. fHbp-1.1:NadA-1.1 and fHbp-2.16:NHBA-20 were very stable, persisting for thirty and forty years, respectively. Combinations generated by fHbp-2.16, NHBA-20 and NadA-3.8 were indicated by the symbol (*). Combinations generated by fHbp-1.1, NHBA-3 and NadA-1.1 were indicated by the symbol (§).</p