ZnuD, a potential candidate for a simple and universal Neisseria meningitidis vaccine

Abstract

Neisseria meningitidis serogroup B (MenB) is a major cause of bacterial sepsis and meningitis, with the highest disease burden in young children. Available vaccines are based on outer membrane vesicles (OMVs) obtained from wild-type strains. However, particularly in toddlers and infants, they confer protection mostly against strains expressing the homologous protein PorA, a major and variable outer membrane protein. In the quest for alternative vaccine antigens able to provide broad MenB strain coverage in younger populations, but potentially also across all age groups, ZnuD, a protein expressed under zinc-limiting conditions, may be considered a promising candidate. Here, we have investigated the potential value of ZnuD and show that it is a conserved antigen expressed by all MenB strains tested except for some strains of clonal complex ST-8. In mice and guinea pigs immunized with ZnuD-expressing OMVs, antibodies were elicited that were able to trigger complement-mediated killing of all the MenB strains and serogroup A, C, and Y strains tested when grown under conditions of zinc limitation. ZnuD is also expressed during infection, since anti-ZnuD antibodies were detected in sera from patients. In conclusion, we confirm the potential of ZnuD-bearing OMVs as a component of an effective MenB vaccine.

Fig 1

Wired-frame representation of the ZnuD 3D model. In this model, computed by Modeler, the 3D structure of extracellular loops at residues 275 to 324 and 636 to 657 was not predicted.

Fig 2

Responses of human sera obtained from convalescent patients (n = 12) (A) and healthy carriers (n = 11) (B) in a ZnuD peptide array. The localization of 11 putative exposed loops is indicated. Box plots indicate ranges, medians, and 25th and 75th percentiles. The dashed line indicates a foreground (FG) intensity-to-background (BG) intensity ratio of 2.

Fig 3

Neighbor-joining tree of ZnuD amino acid sequences analyzed in this study. A total of 765 amino acid positions were analyzed. An unrooted tree is shown. A total of 2,000 bootstrap replications were conducted. The strain designations and clonal complexes are provided as taxon information.

Fig 4

Phylogenetic relationships of ZnuD amino acid sequences of 505 strains isolated in England, Wales, and Northern Ireland within a period of 1 year. The neighbor-joining method was used to analyze the data. The p-distance method was applied. Bootstrapping was used with 2,000 replications.

Fig 5

Entropy plot of ZnuD amino acid sequences of 505 strains isolated in the United Kingdom within a period of 1 year. A total of 758 positions were investigated. Entropy represents the variability or uncertainty at a given position of the sequence. The entropy is “0” if the site is not variable. The x axis provides the amino acid along with the predicted positions of 11 extracellular loops.

Fig 6

(A to D) Growth of wild-type ZnuD-expressing (black lines) and ZnuD-nonexpressing (gray lines) MenB/C ST-8 cc/cluster A4 (A and B) and MenY ST-23 cc (C and D) strains. Bacteria were grown in RPMI medium supplemented with 100 μM ferric chloride in the absence (A and C) or in the presence (B and D) of 1.0 μM TPEN. The OD₆₀₀ of the bacterial culture was determined every 2 h. Each strain was tested in at least two independent experiments. (E) Growth of ZnuD-negative DE8633 complemented either with control vector pAP2-1 (black bars) or with pIK6 (gray bars) after 24 h in RPMI medium supplemented with 100 μM ferric chloride and different concentrations of TPEN. The graph shows the means of three independent experiments with standard deviations.