Pattern recognition receptors in microbial keratitis.

Microbial keratitis is a significant cause of global visual impairment and blindness. Corneal infection can be caused by a wide variety of pathogens, each of which exhibits a range of mechanisms by which the immune system is activated. The complexity of the immune response to corneal infection is only now beginning to be elucidated. Crucial to the cornea's defences are the pattern-recognition receptors: Toll-like and Nod-like receptors and the subsequent activation of inflammatory pathways. These inflammatory pathways include the inflammasome and can lead to significant tissue destruction and corneal damage, with the potential for resultant blindness. Understanding the immune mechanisms behind this tissue destruction may enable improved identification of therapeutic targets to aid development of more specific therapies for reducing corneal damage in infectious keratitis. This review summarises current knowledge of pattern-recognition receptors and their downstream pathways in response to the major keratitis-causing organisms and alludes to potential therapeutic approaches that could alleviate corneal blindness.

Distribution of carried pneumococcal clones in UK children following the introduction of the 7-valent pneumococcal conjugate vaccine: a 3-year cross-sectional population based analysis.

The success of Streptococcus pneumoniae (pneumococcus) in both colonisation and disease is associated with the increased prevalence of genetic clones expressing virulence factors that assist host invasion. We studied the distribution of pneumococcal clones in paediatric carriage as part of an ongoing longitudinal study of pneumococcal carriage in children less than 5 years of age. Across three years, 87 different sequence types (STs) were found amongst 310 pneumococci. A decline in PCV-7 related STs was observed during the study period. STs 62, 199, 433 and 1692 increased after the implementation of PCV-7 and were related to increases in serotypes 11A, 19A, 22F, and to serotype 6C, respectively. Overall, a strong correlation was observed between ST and serotype. Thirteen STs contained multiple serotypes and 74 STs were associated with only one serotype. On-going molecular epidemiological surveillance of pneumococcal carriage is warranted during the implementation of pneumococcal conjugate vaccines.

Effective plasmid DNA and small interfering RNA delivery to diseased human brain microvascular endothelial cells.

Expression of exogenous DNA or small interfering RNA (siRNA) in vitro is significantly affected by the particular delivery system utilized. In this study, we evaluated the transfection efficiency of plasmid DNA and siRNA into human brain microvascular endothelial cells (HBMEC) and meningioma cells, which constitute the blood-cerebrospinal fluid barrier, a target of meningitis-causing pathogens. Chemical transfection methods and various lipofection reagents including Lipofectamin™, FuGene™, or jetPRIME®, as well as physical transfection methods and electroporation techniques were applied. To monitor the transfection efficiencies, HBMEC and meningioma cells were transfected with the reporter plasmid pTagGFP2-actin vector, and efficiency of transfection was estimated by fluorescence microscopy and flow cytometry. We established protocols based on electroporation using Cell Line Nucleofector® Kit V with the Amaxa® Nucleofector® II system from Lonza and the Neon® Transfection system from Invitrogen resulting in up to 41 and 82% green fluorescent protein-positive HBMEC, respectively. Optimal transfection solutions, pulse programs and length were evaluated. We furthermore demonstrated that lipofection is an efficient method to transfect meningioma cells with a transfection efficiency of about 81%. Finally, we applied the successful electroporation protocols to deliver synthetic siRNA to HBMEC and analyzed the role of the actin-binding protein cortactin in Neisseria meningitidis pathogenesis.

Identification of novel pneumolysin alleles from paediatric carriage isolates of Streptococcus pneumoniae.

Pneumolysin (Ply) is a major virulence factor of Streptococcus pneumoniae and is produced by all known clinical isolates of pneumococci. Pneumolysin toxoids are being considered as vaccine candidates. We investigated the diversity of pneumolysin among 194 nasopharyngeal pneumococci characterized by serotyping and multilocus sequence typing (MLST). Eight Ply protein alleles were identified, four of which were novel. The 4 novel alleles varied at 10 different amino acid positions, from a total of 147, 3 of these substitutions have been previously reported in different combinations. The protein allele correlated closely with MLST. It is critical that the presence of pneumolysin variants is considered with regards to the potential use of Ply in future vaccine formulations, as variation in Ply amino acid sequence may influence the immunogenicity of vaccines based on the presence of an individual Ply allele.

Immunization with recombinant Opc outer membrane protein from Neisseria meningitidis: influence of sequence variation and levels of expression on the bactericidal immune response against meningococci.

The opc gene from Neisseria meningitidis was cloned into the pRSETA vector, and recombinant protein was expressed at high levels in Escherichia coli. The protein was readily purified by affinity chromatography and used for immunization with conventional Al(OH)3 adjuvant or after incorporation into liposomes and Zwittergent micelles. The resulting sera were analyzed for their ability to recognize purified recombinant protein and "native" protein in an enzyme immunoassay with outer membranes and by whole-cell immunofluorescence. Immunization with Al(OH)3 induced high levels of antibodies which reacted with the purified protein but did not recognize whole cells. In contrast, liposomes and micelles induced antibodies which reacted with the native protein in whole cells. The addition of monophosphoryl lipid A (MPLA) to either liposomes or micelle preparations increased the magnitude of the immune response and induced a wider range of immunoglobulin subclasses. This was associated with the ability of the sera to induce complement-mediated killing of the homologous strain. The most effective bactericidal activity was observed with Opc protein incorporated into liposomes containing MPLA. The magnitude of the bactericidal effect was strongly influenced by the level of expression of the Opc protein and was abolished by limited variation in the sequence of the protein expressed by heterologous strains.

Interactions of Neisseria gonorrhoeae with mature human macrophage opacity proteins influence production of proinflammatory cytokines.

The pathological features of ascending gonococcal infection suggest that proinflammatory mediators secreted by tissue-resident macrophages are important components of the host response. Challenge of fully differentiated, mature macrophages with variants of Neisseria gonorrhoeae strain P9 or purified bacterial surface components (pili, lipooligosaccharide, and outer membrane vesicles) induced the secretion of interleukin 6 (IL-6), tumor necrosis factor alpha, growth-related protein alpha, macrophage inflammatory protein 1alpha (MIP-1alpha), and RANTES cytokines but had no effect on IL-8 production. No secretion of IL-1beta, epithelial-derived neutrophil attractant 78, granulocyte-macrophage colony-stimulating factor, IL-10, or IL-12 cytokines was observed. Notably, the P9-Opa(b) protein, in comparison to P9-Opa(a), increased the association of gonococci with macrophages and elevated the secretion of cytokines. Thus, variation in Opa protein expression by the gonococcus may be a determining factor in the severity of pelvic inflammatory disease.

Recombinant proteins in vaccine development.

The outer membrane of Neisseria meningitidis contains a variety of proteins with the potential for inclusion in new meningococcal vaccines (1). Studies on the vaccine potential of these proteins would be facilitated by the production of pure recombinant protein, free from other components of the Neisseria outer membrane. At present, the class 1 outer-membrane protein (OMP) is generally regarded as the most promising candidate. In this chapter, we describe four protocols involved in the preparation of recombinant class 1 OMP for vaccine development. This integrated set of methods can also be readily used to study the potential of other meningococcal OMP as vaccine candidates, and moreover, their utilities make them attractive for vaccine studies relating to many other human pathogens.

Epitope mapping.

An epitope is defined as the site on an antigen at which an antibody binds. In the case of proteins the epitopes can be classified as continuous (or sequential) and discontinuous according to whether or not the amino acids recognized are close together in the primary sequence or are well-separated but brought together by the folding of the protein. The methods described have permitted the localization or "mapping" of continuous epitopes on meningococcal outer-membrane proteins (OMPs), which are recognized by both monoclonal and polyclonal antibodies (MAbs/PAbs).

Interactions of Neisseria meningitidis with cells of the human meninges.

The interaction of Neisseria meningitidis with the meninges that surround and protect the brain is a pivotal event in the progression of bacterial meningitis. Two models of the human meninges were established in vitro, using (i) sections of fresh human brain and (ii) cultures of viable cells grown from human meningiomas. Neisseria meningitidis showed a specific predilection for binding to the leptomeninges and meningeal blood vessels in human brain and not to the cerebral cortex. There was a close correlation between the adherence of different Neisseria species to leptomeninges and cultured cells. The major ligand that mediated adherence was the pilus, and pilin variation modulated the interactions. The presence of Opa protein increased the association of Cap+ meningococci that expressed low-adhesive pili, but did not influence the association of high-adhesive pili. In contrast, Opc did not influence the adherence of Cap+ meningococci, whereas loss of capsule was associated with a more intimate interaction between the bacteria and the meningioma cell that was not apparent with Cap+ meningococci. There was no evidence of internalization of meningococci by meningioma cells in vitro, an observation that is consistent with the barrier properties of the leptomeninges to N. meningitidis observed in vivo.

Lack of immunity in university students before an outbreak of serogroup C meningococcal infection.

Immunity to meningococci was determined in infected and uninfected students before and during an outbreak of serogroup C meningococcal infection at a university in the United Kingdom. No immunity against the outbreak strain was detected in serum taken from infected students prior to the outbreak or at the time of admission; bactericidal activity developed during convalescence. Carriage of all strains of serogroup C meningococci in asymptomatic students was low (0.9%), and no carriage of the outbreak strain could be detected. Immunity in the at-risk student population before the outbreak was low: 90% of students had no significant bactericidal activity against the outbreak strain. A low prevalence of carriage of the outbreak strain, together with a low prevalence of protective immunity within the student population, was associated with a high incidence of invasive disease in those who acquired the outbreak strain.

Interaction of primary human endometrial cells with Neisseria gonorrhoeae expressing green fluorescent protein.

Infection of the endometrium by Neisseria gonorrhoeae is a pivotal stage in the development of pelvic inflammatory disease in women. An ex vivo model of cultures of primary human endometrial cells was developed to study gonococcal-host cell interactions. To facilitate these studies, gonococci were transformed with a hybrid shuttle vector containing the gfp gene from Aequoria victoria, encoding the green fluorescent protein (GFP), to produce intrinsically fluorescent bacteria. The model demonstrated that both pili and Opa proteins were important for both mediating gonococcal interactions with endometrial cells and inducing the secretion of pro-inflammatory cytokines and chemokines. Pil+ gonococci showed high levels of adherence and invasion, regardless of Opa expression, which was associated with increased secretion of IL-8 chemokine and reduced secretion of IL-6 cytokine. Gonococcal challenge also caused increased secretion of TNF-alpha cytokine, but this did not correlate with expression of pili or Opa, suggesting that release of components from non-adherent bacteria may be involved in TNF-alpha induction. Thus, the use of cultured primary endometrial cells, together with gonococci expressing green fluorescent protein, has the potential to extend significantly our knowledge, at the molecular level, of the role of this important human pathogen in the immunobiology of pelvic inflammatory disease.

Effect of adjuvant composition on immune response to a multiple antigen peptide (MAP) containing a protective epitope from Neisseria meningitidis class 1 porin.

A variety of adjuvants with the potential for use with experimental human vaccines were used for immunisation of mice, in an attempt to augment the humoral immune response to a multiple antigen peptide (MAP) containing a protective epitope from the sero-subtype specific class 1 porin protein of Neisseria meningitidis, in tandem with a Th-cell epitope. Surface plasmon resonance showed that combinations of the immunomodulators pluronic block co-polymer, muramyl dipeptide and monophosphoryl lipid A (MPL), increased the magnitude and avidity of the immune response in comparison with both Al(OH)3 and Freund-type adjuvants. In addition, the incorporation of MPL was essential for the induction of a broad distribution of antibody isotypes. The antibodies induced recognised the native protein in meningococcal outer membranes in a subtype-specific manner. The formulations containing these multiple immunomodulators which have already been used in human phase I/II trials with experimental vaccines, are candidates for inclusion in future human vaccines based on synthetic peptides containing defined, protective epitopes.

Dynamics of carriage of Neisseria meningitidis in a group of military recruits: subtype stability and specificity of the immune response following colonization.

Meningococcal carriage and the immune response to colonization were studied in a group of military recruits undergoing basic training. Subtyping by determination of the class 1 protein sequence clearly differentiated between strains and demonstrated the dynamics of carriage and transmission. Expression of class 1 protein by each strain remained stable during prolonged carriage by different subjects. Following colonization, a marked increase in serum bactericidal response occurred, which was specific for the subtype of the acquired strain and was associated with an increase in reactivity by Western blot to the homologous class 1 protein. Subjects colonized by multiple strains showed evidence of a specific immune response to the class 1 protein of each strain acquired. The subtype specificity of the bactericidal response to meningococci and the stability of expression of the class 1 protein have important implications for the design of vaccines for prevention of serogroup B meningococcal disease.

Expression of Neisseria meningitidis class 1 porin as a fusion protein in Escherichia coli: the influence of liposomes and adjuvants on the production of a bactericidal immune response.

High level expression of meningococcal class 1 protein was achieved in Escherichia coli using the p-GEMEX-1 vector, in which the protein was expressed in inclusion bodies (IB), as a fusion with the bacteriophage T7 gene 10 capsid protein. The fusion protein (FP) was engineered with a factor Xa protease site between the gene 10 and class 1 protein, but treatment with the enzyme resulted in cleavage at additional sites within the class 1 protein. Since it was not possible to remove the leader protein, the intact FP provided an alternative antigen for immunization. Antisera raised to FP, solubilized from IB and incorporated into liposomes, generated a subtype-specific response which was weakly bactericidal for meningococci. In order to remove any possible effect of E. coli LPS present in IB, the FP was further purified by SDS-PAGE and incorporated into liposomes, either alone or in combination with the adjuvants monophosphoryl lipid A or muramyl dipeptide. The incorporation of adjuvants in liposomes resulted in stimulation of the overall immune response to FP, but the resulting antisera were not bactericidal. However an effective bactericidal response was obtained with the purest preparation of FP in liposomes, without any additional adjuvants, revealing that attempts to increase further the immunogenicity of such antigens must not be at the expense of interfering with optimal protein folding.

Novel trinucleotide deletion in Fabry's disease.

We describe the molecular characterization of a novel, in-frame deletion that is located in exon 7 of the alpha-galactosidase A gene in a patient with Fabry's disease. The 3-bp deletion we identified, besides the typical severe clinical features, also expresses diffuse facial telangiectasias, which is a new cutaneous marker of Fabry's disease.

Immunization with a multiple antigen peptide containing defined B- and T-cell epitopes: production of bactericidal antibodies against group B Neisseria meningitidis.

Previous analysis of the class 1 outer-membrane (OM) protein of Neisseria meningitidis has identified discrete epitopes to be potential targets for immune attack. The conformation of these epitopes is important for inducing antibodies which can react with the native protein and promote complement-mediated lysis of the meningococcus. The multiple antigen peptide (MAP) system, which consists of an oligomeric branching lysine core to which are attached dendritic arms of defined peptide antigens, confers some conformational stability and also allows for the preparation of immunogens containing both B-cell and T helper (Th)-cell epitopes. In this study, MAPs were synthesized to contain (i) the subtype P1.16b meningococcal class 1 protein B-cell epitope (B-MAP), and (ii) the P1.16b epitope in tandem with a defined Th-cell epitope, chosen from tetanus toxin (BT-MAP). The B-MAP was nonimmunogenic in animals. In contrast, incorporation of the Th-cell epitope into BT-MAP induced a strong humoral response towards the class 1 protein B-cell epitope. Antisera from immunized mice and rabbits reacted in ELISA with synthetic peptides containing the B-cell epitope, and also cross-reacted with meningococcal OMs from strains of subtype P1.16b and P1.16a. Murine and rabbit antisera showed similar reactivity and epitope specificity, but did not react with denatured class 1 protein in Western blotting, indicating the predominance of antibodies directed towards conformational epitopes. The antisera from rabbits immunized with BT-MAP promoted complement-mediated bactericidal killing not only of the homologous meningococcal subtype P1.16b strain but also of subtype P1.16a.

Immunization with synthetic peptides containing epitopes of the class 1 outer-membrane protein of Neisseria meningitidis: production of bactericidal antibodies on immunization with a cyclic peptide.

The class 1 outer-membrane protein of Neisseria meningitidis is the target for subtype-specific, bactericidal monoclonal antibodies (mAbs). The epitopes recognized by these antibodies have been mapped previously to linear peptides corresponding to the sequences thought to be exposed at the apices of surface-exposed loops of the protein. In this work several synthetic peptides containing the subtype Pl.16b epitope have been synthetized with the aim of inducing a polyclonal immune response resembling the reactivity of the mAbs. Initially, peptides of 9 and 15 amino acid residues were synthesized and used for immunization after coupling to a carrier protein. The reactivity of the resulting antisera, with synthetic linear decapeptides, resembled that seen in previous epitope mapping experiments with the protective mAbs. However, despite the induction of antibodies having the desired specificity, the antisera reacted poorly with the native protein in outer membranes, and were non-bactericidal. A 36mer peptide, consisting of the entire surface-exposed loop 4 of the class 1 protein was then synthesized and used for immunization as (i) free peptide, (ii) peptide coupled to carrier and (iii) peptide subjected to cyclization, in an attempt to restrict it to conformations that might more closely resemble the native loop structure. In contrast to antisera raised against linear peptides, antibodies raised by immunization with the 36mer cyclic peptide, did not react with linear peptides recognized by the mAbs, but instead appeared to recognize conformational determinants. This antiserum promoted complement-mediated bactericidal killing of the homologous meningococcal strain, demonstrating the potential of synthetic peptide immunogens for inducing a protective immune response against group B meningococci.

The potency of tissue-type plasminogen activator (TPA) determined with chromogen and clot-lysis assays.

An assessment was made of two methods for determining the potency of tissue-type plasminogen activator (TPA). A chromogenic microtitre plate assay was established which contained TPA, plasminogen, a synthetic plasmin substrate (H-D-valyl-L-leucyl-L-lysyl-p-nitroaniline dihydrochloride, S2251) and any one of the following stimulators: native fibrinogen, enzymatic and chemical digests of fibrinogen, poly-D-lysine (PDL) and chemical derivatives of the latter. The chromogen assay was compared with an automated clot-lysis (turbidimetric) assay for sensitivity, reproducibility and validity for potency determination. Reference preparations of TPA were titrated in both assays: in the chromogen assay the dose-response curves were non-parallel, whereas parallelism was observed in the clot-lysis assay. Thus, the chromogen assay was restricted in its applicability and disqualified from any routine regulatory use. The potency of individual lots of recombinant (r)TPA could only be estimated in International Units (IU) of TPA activity with the automated clot-lysis assay and the potency values obtained (IU/vial) were in remarkably close agreement with the manufacturers' values.

Pertussis vaccines: present status.

The eradication of pertussis as a worldwide disease is unforeseeable for the present and immediate future. Mortality and morbidity from clinical pertussis are still commonly reported, especially in underdeveloped countries where mass immunization programs are virtually nonexistent. To achieve control of the disease, a high level of immunization coverage is necessary, and pertussis WCPV still remains one of the most effective bacterial vaccines. But, primarily because of its reactogenicity for infants, much effort has been directed toward the characterization of bacterial components important in pathogenesis and for the development of acellular vaccines. Progress in the last decade has resulted in the production and use of such vaccines for routine vaccination, and their use in Japan, as well as the recent clinical trials in Sweden and several phase I/II studies in other countries, has shown that these preparations are safer than conventional WCPV, and equally effective, in preventing disease. The development of future acellular pertussis vaccines by gene manipulation may finally inspire public confidence for vaccine prophylaxis, eventually leading to eradication of the disease. However, the production and use of such sophisticated vaccines is dependent on many factors, and consequently conventional WCPV may still be used in many countries for several years to come.