Article Figures & Data
Figures
- 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.
Tables
- TABLE 1.
Description and origins of Neisserial strains used in this studya
Strain Serogroup type:subtype 2086 subfamily Source Location (country) 2086 expression on Western blot Phylogenetic cluster M98 250670 B:1:P1.4 B MPHL UK + B8 M98 250024 B:1:P1.4 B MPHL UK + B5 M97 253524 B:1:P1.4 B MPHL UK + B8 M97 252060 B:1:P1.4 B MPHL UK + B5 M97 251870 B:4z:P1.4 B MPHL UK + B5 M97 251854 B:4z:P1.4 A MPHL UK + A1 M97 251836 B:4z:P1.4 B MPHL UK + B5 M97 251830 B:4z:P1.4 B MPHL UK + B5 M97 251905 B:4z:P1.4 B MPHL UK + B5 M97 251898 B:4z:P1.4 B MPHL UK + B5 M97 251885 B:4z:P1.4 B MPHL UK + B8 M97 251876 B:4z:P1.4 B MPHL UK + B5 M97 251994 B:4z:P1.4 B MPHL UK + B5 M97 251985 B:4z:P1.4 B MPHL UK + B5 M97 251957 B:4z:P1.4 B MPHL UK + B5 M97 251926 B:4z:P1.4 B MPHL UK + B8 M97 252045 B:4z:P1.4 B MPHL UK + ND M97 252038 B:4z:P1.4 B MPHL UK + ND M97 252026 B:4z:P1.4 B MPHL UK + ND M97 252010 B:4z:P1.4 B MPHL UK + ND M97 252098 B:4z:P1.4 B MPHL UK + ND M97 252083 B:4z:P1.4 B MPHL UK + ND M97 252078 B:4z:P1.4 B MPHL UK + ND M98 250735 B:4z:P1.15 B MPHL UK + ND M98 250797 B:4z:P1.15 B MPHL UK + ND M98 250768 B:4z:P1.15 B MPHL UK + ND M98 250622 B:2b:P1.10 A MPHL UK + A1 M98 250572 B:2b:P1.10 A MPHL UK + A1 M98 250716 B:2b:P1.10 A MPHL UK + A1 M98 250699 B:4z:P1.10 B MPHL UK + ND M98 250393 B:4z:P1.10 B MPHL UK + ND M98 250173 B:4z:P1.10 B MPHL UK + ND M98 250771 B:4z:P1.22,14 A MPHL UK + A12 M98 250732 B:4z:P1.22,14-1 A MPHL UK + A12 CDC-1343 B:NT:P1.22,14 B CDC US + B1 CDC-2369 B:NT:P1.22-1,14-1 A CDC US + A7 6557 B:7:P1.22-1,14 A RIVM US + A3 M97 253462 B:4z:P1.14 B MPHL UK + ND CDC-1127 B:NT:P1.7,16 B CDC US + ND CDC-982 B4:P1.7,16 B CDC US + ND CDC-1359 B4:P1.7,16 B CDC US + ND M98 250762 B:15:P1.7,16 B MPHL UK + ND M98 250610 B:15:P1.7,16 B MPHL UK + ND M98 250626 B:15:P1.16 B MPHL UK + ND M98 250809 B:15:P1.7,16 A MPHL UK + A2 M97 253248 B:15:P1.7,16 A MPHL UK + A2 M97 252029 B:15:P1.7,16 B MPHL UK + ND M97 251875 B:15:P1.7,16-10 B MPHL UK + ND CDC-798 B:15:P1.7,16 B CDC US + ND CDC-1078 B:15:P1.7,16 B CDC US + ND CDC-1614 B:15:P1.7,16 B CDC US + ND CDC-1658 B:15:P1.7,16 B CDC US + ND CDC-937 B:15:P1.7,16 B CDC US + B4 H44/76 B:15:P1.7,16 B RIVM Norway + B4 M97 250571 B:15:P1.16 B MPHL UK + B4 M97 252097 B:15:P1.16 B MPHL UK + B4 M97 253092 B:1:P1.6 B MPHL UK + ND M97 252697 B:1:P1.18,25,6 A MPHL UK + A3 M97 252988 B:4:P1.18,25,6 A MPHL UK + A3 M97 252976 B:4:P1.18,25,6 A MPHL UK + A1 M97 252153 B:4:P1.18,25,6 A MPHL UK + A11 6940 B:NT:P1.18,25,6 B RIVM US + B2 CDC-1610 B:4:P1.18-7,16-4 A CDC US + A3 CDC-1521 B:2b:P1.6 A CDC US + A1 CDC-1985 B:4:P1.7,13 B CDC US + ND CDC-1034 B:4:P1.7 A CDC US + A5 L6 M992 B:NT:P1.7,1 ND Walter Reed + ND/PICK> L8 M978 B:15:P1.7,1 A Walter Reed + A3 CDC-1492 B:4:P1.7,1 A CDC US + A3 CDC-1573 B:4:P1.7-1,1 B CDC US + B9 L7 6155 B:4:P1.7,1 B Walter Reed + ND 8529 B:15:P1.7-2,3 B RIVM Chile + B4 880049 B:4:P1.7-2,4 B RIVM Netherlands + B8 870446 B:4:P1.12-1,13 A RIVM Netherlands + A2 CDC-2367 B:4:P1.15 B CDC US + B4 H355 B:15:P1.19,15 B RIVM Norway + B4 M982 B:9:P1.22,9 B RIVM US + B6 870227 B:4:P1.5-3,10 B RIVM Netherlands + B4 B40 A:NT:P1.5-3,10 B RIVM + B5 5315 B:4:P1.5-3,10 B RIVM + B5 2996 B:2b:P1.5-1,2-2 A RIVM US + A1 NMB B:2b:P1.5-1,2-2 A UIOWA US + A1 CDC-983 B:2a:P1.5,2 B CDC US + B3 L3 6275 B:2a:P1.5,2 A Walter Reed + A8 CDC-852 B:NT:P1.5,2 B CDC US + B7 B16B6 B:2a:P1.5,2 A RIVM + A4 L4 891 C:NT:P1.21,16 A Walter Reed + A6 L5 M981 B:4:P1.21-6,1 A Walter Reed + A1 CDC-1135 B:NT A CDC US + A9 A4 A:NT B + B5 C11 C:16,P1.7-1,1 A CDC Germany + A4 Y Y: A Walter Reed + A4 W135 W135: A Walter Reed + A4 Ng-FA1090 A UNC US + A10 Nl-UR5 A UR + ND Ns-UR4 — UR − ND ↵a Strains are arbitrarily arranged by PorA serosubtype. The 2086 gene subfamily was determined by PCR screening as described in Materials and Methods. A dash indicates negative results; ND indicates not done. Country or origin of each neisserial isolate is listed where known. UK, United Kingdom; US, United States. The 2086 gene was sequenced from those strains where a phylogenetic cluster number based on amino acid sequence homology is indicated (see Fig. 1).
- TABLE 2.
Primers used in PCR amplification experiments
Primer name Sequence Restriction site (underlined) 2086NDE 5′-CTATTCTGCATATGACTAGGAGC-3′ NdeI 3STP2086 5′-GCGCGGATCCTTACTGCTTGGCGGCAAGACC-3′ BamHI 5UNI2086 5′-CTATTCTGCGTATGACTAG-3′ NAa 3UNI2086 5′-GTCCGAACGGTAAATTATCGTG-3′ NA 5INT2086 5′-TGCCGATGCACTAACCGCACC-3′ NA 3INT2086 5′-CGTTTCGCAACCATCTTCCCG-3′ NA 5OUT2086 5′-GACAGCCTGATAAACC-3′ NA 3OUT2086 5′-GATGCCGATTTCGTGAAC-3′ NA N2086ILE 5′-GCGCAGATCTCATATGAGCAGCGGAGGGGGTGGTGTCGCCGCCGAYATWGGTGCGGGGCTTGCCG-3′ BglII/NdeI 20863STP 5′-GAGATCTCACTCACTCATTACTGCTTGGCGGCAAGACCGATATG-3′ BglII N1573PCR 5′-GAGATCTCATATGAGCAGCGGAGGCGGCGGAAGC-3′ BglII N29963STP 5′-GAGATCTCACTCACTCACTACTGTTTGCCGGCGATGCCGATTTC-3′ BglII 5LIP2086 5′-GCGGATCCAGCGGAGGGGGTGGTGTCGCC-3′ BamHI 3SPH2086 5′-GCGCATGCTTACTGCTTGGCGGCAAGACCGATATG-3′ SphI 5LIP1573 5′-GCGGATCCAGCGGAGGCGGCGGAAGC-3′ BamHI 5LIP2996 5′-GCGGATCCAGCGGAGGCGGCGGTGTCGCC-3′ BamHI N2996SPH 5′-GCGCATGCCTACTGTTTGCCGGCGATG-3′ SphI 5LIP771 5′-GCGGATCCAGCGGAAGCGGAAGCGGAGGCGGCGGTGTCGCC-3′ BamHI PorABg12Fwd 5′-CGCGAGATCTCATATGGATGTCAGCCTATACGGCGAAATCAAAGCCGGCGTGGAAGGCAGGAACTACCAG-3′ BglII/NdeI PorABg12Ter 5′-CGCGAGATCTTCAGTCACTCATTAGAATTTGTGGCGCAAACCGACG-3′ BglII ↵a NA, not applicable.
- TABLE 3.
Whole-cell ELISA and bactericidal titers of mouse antibodies raised against rLP2086 tested against multiple serogroup B meningococcal strains
Test strain Test strain serosubtype rLP2086-8529 anti- sera (subfamily B) rLP2086-2996 anti- sera (subfamily A) Whole-cell ELISA titera BC50 titerb Whole-cell ELISA titera BC50 titerb Subfamily B 539 P1.7-2,3 >1,458,000 3,200 — — H44/76 P1.7,16 >1,458,000 3,200 56,386 <25 H355 P1.19,15 >1,458,000 3,200 — — CDC-937 P1.7-2,3-4 >1,458,000 >800 — — M97 252097 P1.7-2,16 >1,458,000 >800 — — 6940 P1.18,25,6 900,162 >800 11,157 — M982 P1.22,9 435,909 200 6,424 — 880049 P1.7-2,4 349,912 400 10,588 — CDC-1573 P1.7-1,1 102,508 25 7,871 — Subfamily A 870446 P1.12-1,13 389,829 800 >1,458,000 >800 M98 250771 P1.22,14 139,397 <25 447,867 800 M98 250732 P1.22,14-1 23,928 — 241,510 >800 M97 252697 P1.18,25,6 99,580 — 320,732 >800 6557 P1.22-1,14 25,590 <25 77,319 <25 NMB P1.5-1,2-2 <2,000 <25 8,979 <25 ↵a Endpoint titers expressed as the reciprocal of the dilution at absorbance = 0.1.
↵b Bactericidal (BC50) titers are represented as the reciprocal of the dilution of antiserum that reduced viable cell count by 50%. Week 0 normal mouse sera had BC50 titers of <25. A dash indicates samples were not tested.
- TABLE 4.
rLP2086 elicits greater bactericidal antibody than rP2086 in mice
Antigen Test strain titera H44/76 M97 252697 rP2086-8529 (nonlipidated) 200 — rLP2086-8529 (lipidated) 3,200 — rP2086-1573 (nonlipidated) <25 — rLP2086-1573 (lipidated) 200 — rP2086-2996 (nonlipidated) — <25 rLP2086-2996 (lipidated) — >800 ↵a Bactericidal (BC50) titers represented as the reciprocal of the dilution of antiserum that reduced the viable cell count by 50%. Week 0 normal mouse serum had BC50 titers of <50. A dash indicates samples were not tested.
- TABLE 5.
Purified rLP2086 from subfamily A or subfamily B elicits bactericidal antibodies in mice which are bactericidal against several strains of serogroup B N. meningitidis
Antigen Titera Subfamily B test strains Subfamily A test strains H44/76 880049 1573 6940 M982 870446 M98 250771 M98 250732 M97 252697 Subfamily B rLP2086-8529 3,200 400 <25 >800 200 800 <25 — — rLP2086-880049 400 >800 100 400 200 400 — — — rLP2086-1573 200 >800 800 200 100 50 — — — rLP2086-M982 >800 >800 <25 200 >800 400 — — — Subfamily A rLP2086-2996 <25 — — — — >800 800 >800 >800 rLP2086-870446 — — — — — >800 400 400 400 rLP2086-250771 — — — — — >800 >800 >800 >800 rLP2086-252988 — — — — — >800 >800 >800 >800 rLP2086-C11 — — — — — >800 200 200 400 ↵a Bactericidal (BC50) titers represented as the reciprocal of the dilution of antiserum that reduced viable cell count by 50%. Week 0 normal mouse sera had BC50 titers of <50. A dash indicates samples were not tested.
- TABLE 6.
Antisera from mice immunized with mixtures of rLP2086 variants and rPorA are bactericidal against multiple strains of serogroup B meningococcal strains
Test strain Bactericidal titer rLP2086 monovalent controla rPorA monovalent controlb rLP2086-8529 + rLP2086-2996 rLP2086-8529 + rLP2086-2996 + rP1.22-1,14 + rP1.5-1,2-2 H44/76 >800 — >800 >800 6940 >800 — >800 800 880049 200 — 200 100 M982 400 — 400 200 870446 >800 — >800 >800 M98 250771 800 — 800 400 M98 250732 >800 — >800 400 M97 252697 >800 — >800 >800 6557 <25 800 <25 200 NMB <25 >800 — >800 ↵a The monovalent control for subfamily B strains H44/76, 6940, 880049, and M982 was mouse antiserum raised against rLP2086-8529, and the monovalent control for subfamily A strains 870446, M98 250771, M98 250732, M97 252697, 6557, and NMB was mouse antiserum raised against rLP2086-2996.
↵b The rPorA monovalent control for strain 6557 was mouse antiserum raised against rP1.22-1,14-1, and the monovalent control for NMB was mouse antiserum raised against rP1.5-1,2-2. A dash indicates samples were not tested.
