Vaccine potential of the Neisseria meningitidis 2086 lipoprotein

Abstract

A novel antigen that induces cross-reactive bactericidal antibodies against a number of Neisseria meningitidis strains is described. This antigen, a approximately 28-kDa lipoprotein called LP2086, was first observed within a complex mixture of soluble outer membrane proteins (sOMPs) following a series of fractionation, protein purification, and proteomics steps. Approximately 95 different neisserial isolates tested positive by Western blotting and PCR screening methods for the presence of the protein and the gene encoding LP2086. The strains tested included isolates of N. meningitidis serogroups A, B, C, W135, and Y, Neisseria gonorrhoeae, and Neisseria lactamica. To better understand the microheterogeneity of this protein, the 2086 genes from 63 neisserial isolates were sequenced. Two different subfamilies of LP2086 were identified based on deduced amino acid sequence homology. A high degree of amino acid sequence similarity exists within each 2086 subfamily. The highest degree of genetic diversity was seen between the two subfamilies which share approximately 60 to 75% homology at the nucleic acid level. Flow cytometry (fluorescence-activated cell sorting) analyses and electron microscopy indicated that the LP2086 is localized on the outer surface of N. meningitidis. Antiserum produced against a single protein variant was capable of eliciting bactericidal activity against strains expressing different serosubtype antigens. Combining one recombinant lipidated 2086 (rLP2086) variant from each subfamily with two rPorA variants elicited bactericidal activity against all strains tested. The rLP2086 family of antigens are candidates worthy of further vaccine development.

FIG. 1.

Phylogenetic tree showing the strain clustering according to 2086 protein distances. Subfamily A and B grouping is indicated on the main branches. The tree was constructed with LASERGENE software using ClustalW and was based on mature protein sequences of 254 to 262 amino acids in length. One representative strain and the respective group, type, and serosubtype from each cluster are shown. Boxes indicate 2086 strains chosen for expression studies. The group, type, and subtype of each neisserial isolate is indicated where available. Cluster identity for each of the 63 neisserial 2086 genes sequenced is listed in Table 1. Numbers in square brackets indicate the number of strains with ≥99.6% sequence identity present in each branch of the tree.

FIG. 2.

Comparison of neisserial 2086 sequences. The amino acid sequences of 21 unique 2086 proteins were deduced from the nucleotide sequences and aligned with ClustalW. Dashes indicate gaps. Identical amino acid sequences are indicated by asterisks.

FIG. 3.

Coomassie-stained gel after SDS-PAGE (10 to 20% gradient polyacrylamide). Lane 1, purified rLP2086-8529; lane 2, purified rLP2086-2996.

FIG. 4.

Electron micrographs of N. meningitidis serogroup B strain H44/76 showing a whole cell. (A) Negative control; (B) immunogold labeling with rLP2086-8529-derived antiserum. Bar, 100 nm.

FIG. 5.

FACS analysis of wild-type and knockout strains of N. meningitidis serogroup B labeled with homologous antiserum to rLP2086. (A) Labeling of three wild-type strains with homologous rLP2086 antiserum, with the negative control (week 0) shown as the light gray histogram and the positive antiserum (week 6) shown as a bold unfilled histogram. (B) Labeling of the knockout strains with the same antiserum and the same labels as for panel A, with a light gray histogram for the negative control and a bold line for the anti-rLP2086 serum. (C) Labeling of wild-type (dark gray, filled histogram) and knockout strains (bold unfilled histogram) with the homologous PorA monoclonal antibody specific for each serosubtype.