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

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

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

Distribution of antigens from 1960 to 2008-2009.

<p><b>A) fHbp variants and sub/variants.</b> Out of the 45 sub/variants in frame identified from 1960 to 2008-2009, fHbp-1.14 (16%), 2.16 (13%), 1.1 (8%), 1.4 (8%), and 2.24 (8%) were the most frequent and persisting. fHbp-2.24 was predominant in 1960, 2.16 in 1970, 1.4 in 1980, 1.14 in 1990, 2000 and 2008-2009, together with 1.1. <b>B) NHBA peptides.</b> Out of the 43 peptides identified from 1960 to 2008-2009, NHBA- 2 (24%) and 20 (22%) were the most frequent and persisting. NHBA-2 was predominant in 1960, 1990, 2000 and 2008-2009, NHBA-20 in 1970 and 1980 (this latter case, together with NHBA-29). <b>C) NadA presence, variants and sub/variants. </b><i>nadA</i> gene was absent or presented frame-shift mutations or insertion sequences in 115 out of 165 isolates. Out of the 11 sub/variants identified from 1960 to 2008-2009 in the 50 isolates with the gene, NadA-3.8 (40%) and NadA-1.1 (28%) were the most frequent and persisting. NadA-3.8 was present from 1960 to 1990, NadA-1.1 from 1980 to 2008-2009.</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

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

Distribution of the three fHbp variants Over Time.

<p>fHbp-2 predominated in 1960-1980 and was subsequently substituted by fHbp-1. The presence of fHbp-3 in 2008-2009 was mainly related with the emergence of the cc213.</p

Distribution of the MLST clonal complexes from 1960 to 2008-2009 in the Netherlands.

<p>Clonal complexes (cc)1, cc11, cc32, cc231, cc254, cc269, cc334, cc41/44 and cc8 persisted for more than two decades. cc41/44 (27%), cc8 (13%), cc11(9%), cc32(8%) and cc269 (7%) were the most frequent. cc41/44, which was present in all time-periods considered, was predominant in 1960, 1990, 2000 and 2008-2009, cc8 was predominant in 1970 and cc1 in 1980. cc5, cc22, cc35, cc37, cc60, cc103, cc162, cc167, cc213, cc1157 were observed only once, cc18, cc364, cc461 were present twice. Over time, there were several ccs that disappeared, such as cc37 and cc167,which were present in 1960 only, and on the other new ccs emerged.</p