Fatal Nongroupable Neisseria meningitidis Disease in Vaccinated Patient Receiving Eculizumab.

Patients receiving eculizumab have an increased risk for meningococcal disease, but most reported cases are attributable to encapsulated meningococcal strains. We describe a case in which a nongroupable meningococcal strain, which rarely causes disease in healthy persons, caused fatal disease in an eculizumab recipient despite meningococcal vaccination.

Eculizumab treatment and impaired opsonophagocytic killing of meningococci by whole blood from immunized adults.

Eculizumab, a humanized anti-complement C5 monoclonal antibody (mAb) for treatment of paroxysmal nocturnal hemoglobinuria (PNH) and atypical hemolytic uremic syndrome, blocks the terminal complement pathway required for serum bactericidal activity (SBA). Because treated patients are at >1000-fold increased risk of meningococcal disease, vaccination is recommended; whether vaccination can protect by opsonophagocytic activity in the absence of SBA is not known. Meningococci were added to anticoagulated blood from 12 healthy adults vaccinated with meningococcal serogroup B and serogroup A, C, W, Y vaccines. Bacterial survival was measured after 3-hour incubation in the presence of eculizumab or control complement factor D inhibitor ACH-4471, which blocks the complement alternative pathway (AP) and is in phase 2 development for treatment of PNH. In the absence of inhibitors, colony formation units (CFUs) per milliliter in blood from all 12 immunized subjects decreased from ∼4000 at time 0 to sterile cultures at 3 hours. In the presence of eculizumab, there was a >22-fold increase in geometric mean CFUs per milliliter (90 596 and 114 683 CFU/mL for serogroup B and C strains, respectively; P < .0001 compared with time 0). In the presence of ACH-4471, there was a >12-fold decrease (23 and 331 CFU/mL, respectively; P < .0001). The lack of meningococci killing by blood containing eculizumab resulted from inhibition of release of C5a, a C5 split product needed for upregulation of phagocytosis. The results provide an explanation for the large number of cases of meningococcal disease in immunized patients being treated with eculizumab and suggest that vaccination may provide better protection against meningococcal disease in patients treated with an AP-specific inhibitor.

Meningococcal Factor H Binding Protein Vaccine Antigens with Increased Thermal Stability and Decreased Binding of Human Factor H.

Neisseria meningitidis causes cases of bacterial meningitis and sepsis. Factor H binding protein (FHbp) is a component of two licensed meningococcal serogroup B vaccines. FHbp recruits the complement regulator factor H (FH) to the bacterial surface, which inhibits the complement alternative pathway and promotes immune evasion. Binding of human FH impairs the protective antibody responses to FHbp, and mutation of FHbp to decrease binding of FH can increase the protective responses. In a previous study, we identified two amino acid substitutions in FHbp variant group 2 that increased its thermal stability by 21°C and stabilized epitopes recognized by protective monoclonal antibodies (MAbs). Our hypothesis was that combining substitutions to increase stability and decrease FH binding would increase protective antibody responses in the presence of human FH. In the present study, we generated four new FHbp single mutants that decreased FH binding and retained binding of anti-FHbp MAbs and immunogenicity in wild-type mice. From these mutants, we selected two, K219N and G220S, to combine with the stabilized double-mutant FHbp antigen. The two triple mutants decreased FH binding >200-fold, increased the thermal stability of the N-terminal domain by 21°C, and bound better to an anti-FHbp MAb than the wild-type FHbp. In human-FH-transgenic mice, the FHbp triple mutants elicited 8- to 15-fold-higher protective antibody responses than the wild-type FHbp antigen. Collectively, the data suggest that mutations to eliminate binding of human FH and to promote conformational stability act synergistically to optimize FHbp immunogenicity.

A meningococcal vaccine antigen engineered to increase thermal stability and stabilize protective epitopes.

Factor H binding protein (FHbp) is part of two vaccines recently licensed for prevention of sepsis and meningitis caused by serogroup B meningococci. FHbp is classified in three phylogenic variant groups that have limited antigenic cross-reactivity, and FHbp variants in one of the groups have low thermal stability. In the present study, we replaced two amino acid residues, R130 and D133, in a stable FHbp variant with their counterparts (L and G) from a less stable variant. The single and double mutants decreased thermal stability of the amino- (N-) terminal domain compared with the wild-type protein as measured by scanning calorimetry. We introduced the converse substitutions, L130R and G133D, in a less stable wild-type FHbp variant, which increased the transition midpoint (Tm) for the N-terminal domain by 8 and 12 °C; together the substitutions increased the Tm by 21 °C. We determined the crystal structure of the double mutant FHbp to 1.6 Å resolution, which showed that R130 and D133 mediated multiple electrostatic interactions. Monoclonal antibodies specific for FHbp epitopes in the N-terminal domain had higher binding affinity for the recombinant double mutant by surface plasmon resonance and to the mutant expressed on meningococci by flow cytometry. The double mutant also had decreased binding of human complement Factor H, which in previous studies increased the protective antibody responses. The stabilized mutant FHbp thus has the potential to stabilize protective epitopes and increase the protective antibody responses to recombinant FHbp vaccines or native outer membrane vesicle vaccines with overexpressed FHbp.

A Newly-Identified Polymorphism in Rhesus Macaque Complement Factor H Modulates Binding Affinity for Meningococcal FHbp.

BACKGROUND: Two meningococcal serogroup B vaccines contain Factor H binding protein (FHbp). Binding of Factor H (FH) to FHbp was thought to be specific for human or chimpanzee FH. However, in a previous study an amino acid polymorphism in rhesus macaque FH domain 6, tyrosine at position 352 (Y352) was associated with high binding to FHbp, whereas histidine at position 352 (H352) was associated with low binding. METHODS AND RESULTS: Here we report that a second FH polymorphism at position 360 also affects macaque FH binding. Of 43 macaques, 11 had high FH binding and 32 had low binding. As in our previous study, all 11 animals with high binding had Y352, and 24 with low binding had H352. However the remaining eight with low FH binding had Y352, which was predicted to yield high binding. All eight had S360 instead of P360. Thus, three allelic variants at positions 352 and 360 affect macaque FH binding to FHbp: HP (low), YS (low), and YP (high). We measured binding affinity of each FH sequence type to FHbp by surface plasmon resonance. Two animals with high binding types (YS/YP and HP/YP) had dissociation constants (KD) of 10.4 and 18.2 nM, respectively, which were similar to human FH (19.8 nM). Two macaques with low binding (HP/HP and HP/YS) had KD values approximately five-fold higher (100.3 and 99.5 nM, respectively). A third macaque with low binding (YS/YS) had a KD value too high to be measured. CONCLUSIONS: Macaques have at least three allelic variants encoding FH with different affinities for FHbp (five genotypic combinations of these variants). Since in previous studies binding of FH to FHbp vaccines decreased protective antibody responses, our data will aid in selection of macaques with FH binding that is similar to humans for further investigation of FHbp vaccine immunogenicity.

A Mutant Library Approach to Identify Improved Meningococcal Factor H Binding Protein Vaccine Antigens.

Factor H binding protein (FHbp) is a virulence factor used by meningococci to evade the host complement system. FHbp elicits bactericidal antibodies in humans and is part of two recently licensed vaccines. Using human complement Factor H (FH) transgenic mice, we previously showed that binding of FH decreased the protective antibody responses to FHbp vaccination. Therefore, in the present study we devised a library-based method to identify mutant FHbp antigens with very low binding of FH. Using an FHbp sequence variant in one of the two licensed vaccines, we displayed an error-prone PCR mutant FHbp library on the surface of Escherichia coli. We used fluorescence-activated cell sorting to isolate FHbp mutants with very low binding of human FH and preserved binding of control anti-FHbp monoclonal antibodies. We sequenced the gene encoding FHbp from selected clones and introduced the mutations into a soluble FHbp construct. Using this approach, we identified several new mutant FHbp vaccine antigens that had very low binding of FH as measured by ELISA and surface plasmon resonance. The new mutant FHbp antigens elicited protective antibody responses in human FH transgenic mice that were up to 20-fold higher than those elicited by the wild-type FHbp antigen. This approach offers the potential to discover mutant antigens that might not be predictable even with protein structural information and potentially can be applied to other microbial vaccine antigens that bind host proteins.

Binding of Complement Factor H (FH) Decreases Protective Anti-FH Binding Protein Antibody Responses of Infant Rhesus Macaques Immunized With a Meningococcal Serogroup B Vaccine.

BACKGROUND: The meningococcal vaccine antigen, factor H (FH)-binding protein (FHbp), binds human complement FH. In human FH transgenic mice, binding decreased protective antibody responses. METHODS: To investigate the effect of primate FH binding, we immunized rhesus macaques with a 4-component serogroup B vaccine (4CMenB). Serum FH in 6 animals bound strongly to FHbp (FHbp-FH(high)) and, in 6 animals, bound weakly to FHbp (FHbp-FH(low)). RESULTS: There were no significant differences between the respective serum bactericidal responses of the 2 groups against meningococcal strains susceptible to antibody to the NadA or PorA vaccine antigens. In contrast, anti-FHbp bactericidal titers were 2-fold lower in FHbp-FH(high) macaques against a strain with an exact FHbp match to the vaccine (P = .08) and were ≥4-fold lower against 4 mutants with other FHbp sequence variants (P ≤ .005, compared with FHbp-FH(low) macaques). Unexpectedly, postimmunization sera from all 12 macaques enhanced FH binding to meningococci. In contrast, serum anti-FHbp antibodies elicited by 4CMenB in mice whose mouse FH did not bind to the vaccine antigen inhibited FH binding. CONCLUSIONS: Binding of FH to FHbp decreases protective anti-FHbp antibody responses of macaques to 4CMenB. Even low levels of FH binding skew the antibody repertoire to FHbp epitopes outside of the FH-binding site, which enhance FH binding.

Importance of inhibition of binding of complement factor H for serum bactericidal antibody responses to meningococcal factor H-binding protein vaccines.

BACKGROUND: Factor H (fH) binding protein (fHbp) is part of vaccines developed for prevention of meningococcal serogroup B disease. More than 610 fHbp amino acid sequence variants have been identified, which can be classified into 2 subfamilies. The extent of cross-protection within a subfamily has been difficult to assess because of strain variation in fHbp expression. METHODS: Using isogenic mutant strains, we compared cross-protective serum antibody responses of mice immunized with 7 divergent fHbp variants in subfamily B, including identification numbers (ID) 1 and 55, which were chosen for vaccine development. RESULTS AND CONCLUSIONS: In the presence of the human complement downregulator fH, the ability of the anti-fHbp antibodies to deposit sufficient complement C3b on the bacterial surface to elicit bactericidal activity required inhibition of binding of fH by the anti-fHbp antibodies. With less bound fH, the bacteria became more susceptible to complement-mediated bactericidal activity. Among the different fHbp sequence variants, those more central in a phylogenic network than ID 1 or 55 elicited anti-fHbp antibodies with broader inhibition of fH binding and broader bactericidal activity. Thus, the more central variants show promise of extending protection to strains with divergent fHbp sequences that are covered poorly by fHbp variants in clinical development.

WhiB2/Rv3260c, a cell division-associated protein of Mycobacterium tuberculosis H37Rv, has properties of a chaperone.

whiB-like genes have been found in all actinomycetes sequenced so far. The amino-acid sequences of WhiB proteins of Mycobacterium tuberculosis H37Rv are highly conserved and participate in several cellular functions. Unlike other WhiB proteins of M. tuberculosis that have properties of protein disulfide reductases, WhiB2 showed properties like a chaperone as it suppressed the aggregation of several model substrates (e.g. citrate synthase, rhodanese and luciferase). Suppression of aggregation of the model substrates did not require ATP. Four cysteine residues of WhiB2 form two intramolecular disulfide bonds; however, chaperone function was unaffected by the redox state of the cysteines. WhiB2 also restored the activity of chemically denatured citrate synthase and did not require either ATP or a co-chaperone for refolding. The results indicate that WhiB2, which has been shown to be associated with cell division in mycobacteria and streptomyces, has evolved independently of other WhiBs, although it retains basic properties of this group of proteins. This is the first report to show that a WhiB protein has chaperone-like function; therefore, this report will have major implications in attempts to understand the role of WhiB proteins in mycobacteria, particularly in cell division.

Identification of key proteins of cultured human intestinal cells involved in interaction with enteroaggregative Escherichia coli.

Enteroaggregative Escherichia coli (EAEC) adherence to human intestinal tissue is known to be mediated by aggregative adherence fimbriae (AAF). However, the host cell molecules involved in EAEC adherence remain uncharacterized. In the present study, four key membrane glycoproteins of cultured human intestinal epithelial cells (INT-407) were found to be involved in the interaction with the T8 strain of EAEC. Nine membrane proteins of INT-407 cells were found to interact with EAEC-T8, of which four were identified as fibronectin, epidermal growth factor receptor (EGFR), Thrombospondin-1 (TSP1) and glucose-regulated protein (GRP-94). Our findings were validated by Western blot using antibody against each identified protein. The adherence of EAEC-T8 to INT-407 cells was reduced to c. 26, 29, 37 and 76% in the presence of the antibody against GRP-94, EGFR, fibronectin and TSP-1, respectively. These findings were further substantiated by flow cytometry, where the final mean fluorescence intensity value of the INT-407 cells (c. 1075) resulting from adherent-labelled bacteria was found to be reduced to c. 26, 132, 228 and 597 in the presence of antibody against GRP-94, EGFR, fibronectin and TSP-1. We propose that GRP-94, EGFR, fibronectin and TSP-1 are involved in the aggregative adherence of EAEC-T8 to INT-407 cells.

Enteroaggregative Escherichia coli induced increase in intracellular calcium concentration modulates cytoskeletal F-actin rearrangement and bacterial entry in INT-407 cells.

BACKGROUND: Enteroaggregative Escherichia coli (EAEC) is an emerging enteric pathogen, associated with cases of acute and persistent diarrhoea worldwide. The pathogenesis of EAEC is yet to be understood. In intestinal epithelium, an increase in [Ca²âº](i) has been attributed due to the action of different enteric pathogens. EAEC was shown to increase [Ca²âº](i) in HEp-2 cells.The present study was undertaken to investigate the effect of EAEC induced increase in [Ca²âº](i) oncultured human intestinal epithelial cells. METHODS: INT-407 cells were infected with EAEC (T8 strain) in the absence and presence of dantrolene (inhibitor of release of Ca²âº from intracellular stores)/verapamil (L-type Ca²âº channel blocker)/BAPTAAM (Ca²âº chelator)/U73122 (PLC inhibitor)/Cytochalasin-D (inhibitor of actin polymerization). [Ca²âº](i) was estimated using Fura-2/AM. Cytoskeletal rearrangement was assessed by F-actin staining using TRITC-phalloidin. The invasiveness of EAEC-T8 to INT-407 cells was checked by electron microscopy and invasion assay. RESULTS: A significant increase in [Ca²âº](i) was observed in EAEC-T8 infected INT-407 cells, which was reduced in presence of dantrolene/verapamil/U73122. EAEC-T8 could induce cytoskeletal F-actin polymerization in INT-407 cells and was found to be invasive in nature. The cytoskeletal rearrangement as well as invasion of EAEC-T8 was attenuated in presence of U73122/dantrolene/BAPTA-AM/verapamil/cytochalasin D. CONCLUSIONS: EAEC induced increase in [Ca²âº](i) seems to play a major role in host cytoskeletal F-actin rearrangements leading to invasion of the organism. GENERAL SIGNIFICANCE: Our study undoubtedly will lead to an improved understanding of EAEC-pathogenesis.

Galactose specific adhesin of enteroaggregative E. coli induces IL-8 secretion via activation of MAPK and STAT-3 in INT-407 cells.

BACKGROUND: Enteroaggregative Escherichia coli (EAEC) is one of the most common bacterial pathogens associated with the etiology of persistent diarrhea. A characteristic feature of EAEC-pathogenesis is the induction of profound inflammatory response in the intestinal epithelium. The present study was designed to investigate the underlying mechanism of inflammatory responses induced by a novel galactose specific adhesin of T7 strain of EAEC (EAEC-T7) in human intestinal epithelial cell line (INT-407). METHODS: INT-407 cells were stimulated with the adhesin in the absence and presence of anti-adhesin (IgG(AD))/d-galactose/H7/staurosporin (inhibitor of PKC)/PD098059 (inhibitor of MEK)/SB203580 (inhibitor of p38-MAPkinase)/AG490 (inhibitor of JAK (-2,-3)/STAT-3 pathway). The expression of activated Raf-1, MEK-1, ERK1/2, JNK, p38-MAPK and STAT-3 was analyzed by Western immunoblot. Release of interleukin-8 (IL-8) was measured by ELISA. RESULTS: The adhesin was found to induce activation of Raf-1, MEK-1, ERK1/2, p38-MAPK and STAT-3, which was reduced in the presence of IgG(AD)/d-galactose. The activation of Raf-1 was found to be attenuated in the presence of H7/staurosporin. The expression of phosphorylated STAT-3 was downregulated in the presence of AG490 and PD098059. Further, the adhesin induced IL-8 secretion was reduced in the presence of the inhibitors of MEK (PD098059), p38-MAPK (SB203580) and JAK (-2,-3)/STAT-3 pathway (AG490). CONCLUSIONS: We propose that STAT-3 activation is quintessential for the galactose specific adhesin induced IL-8 secretion by INT-407 cells and must occur in concert with the activation of ERK1/2. GENERAL SIGNIFICANCE: Our contribution regarding the galactose specific adhesin mediated signaling leads to an improved understanding of the EAEC-pathogenesis and may provide novel therapeutic approaches to combat EAEC infection.

Enteroaggregative Escherichia coli infection induces IL-8 production via activation of mitogen-activated protein kinases and the transcription factors NF-kappaB and AP-1 in INT-407 cells.

Enteroaggregative Escherichia coli (EAEC) is emerging as a cause of acute and persistent diarrhea in developing countries. An important feature of EAEC pathogenesis is the induction of profound inflammatory response in the intestinal epithelium. In this article, we have shown that EAEC-induced activation of mitogen-activated protein kinases (MAPK) (ERK-1/2, JNK and p38MAPK) in cultured human intestinal epithelial cells (INT-407) leads to the induction of DNA-binding activity of NF-kappaB and AP-1, resulting in IL-8 production. Plasmid-cured EAEC could also activate the MAPK and the transcription factors leading to IL-8 secretion, but to a lesser extent than that of wild-type EAEC. Further, pretreatment of these cells with the highly specific MEK inhibitor (PD 098059), the JNK inhibitor (SP 600125), and the p38MAPK inhibitor (SB 203580) resulted in inhibition of the IL-8 secretion by EAEC (wild type as well as plasmid cured)-infected INT-407 cells. These findings demonstrate that the inflammatory response induced by EAEC may be due to the specific stimulation of MAPK signaling pathways leading to nuclear responses. To our knowledge, this is the first article regarding the detailed mechanism of IL-8 secretion from the EAEC-infected human intestinal epithelial cell line.