The maintenance of strain structure in populations of recombining infectious agents.

Using mathematical models that combine population genetic and epidemiological processes, we resolve the paradox that many important pathogens appear to persist as discrete strains despite the constant exchange of genetic material. We show that dominant polymorphic determinants (that is, those that elicit the most effective immune responses) will be organized into nonoverlapping combinations as a result of selection by the host immune system, thereby defining a set of discrete independently transmitted strains. By analysing 222 isolates of Neisseria meningitidis, we show that two highly polymorphic epitopes of the outer membrane protein PorA exist in nonoverlapping combinations as predicted by this general framework. The model indicates that dominant polymorphic determinants will be in linkage disequilibrium, despite frequent genetic exchange, even though they may be encoded by several unlinked genes. This suggests that the detection of nonrandom associations between epitope regions can be employed as a novel strategem for identifying dominant polymorphic antigens.

Crystal structure of an Fab fragment in complex with a meningococcal serosubtype antigen and a protein G domain.

Many pathogens present highly variable surface proteins to their host as a means of evading immune responses. The structure of a peptide antigen corresponding to the subtype P1.7 variant of the porin PorA from the human pathogen Neisseria meningitidis was determined by solution of the X-ray crystal structure of the ternary complex of the peptide (ANGGASGQVK) in complex with a Fab fragment and a domain from streptococcal protein G to 1.95 A resolution. The peptide adopted a beta-hairpin structure with a type I beta-turn between residues Gly4P and Gly7P, the conformation of the peptide being further stabilised by a pair of hydrogen bonds from the side-chain of Asn2P to main-chain atoms in Val9P. The antigen binding site within the Fab formed a distinct crevice lined by a high proportion of apolar amino acids. Recognition was supplemented by hydrogen bonds from heavy chain residues Thr50H, Asp95H, Leu97H and Tyr100H to main-chain and side-chain atoms in the peptide. Complementarity-determining region (CDR) 3 of the heavy chain was responsible for approximately 50 % of the buried surface area formed by peptide-Fab binding, with the remainder made up from CDRs 1 and 3 of the light chain and CDRs 1 and 2 of the heavy chain. Knowledge of the structures of variable surface antigens such as PorA is an essential prerequisite to a molecular understanding of antigenic variation and its implications for vaccine design.

A novel meningococcal outer membrane vesicle vaccine with constitutive expression of FetA: A phase I clinical trial.

OBJECTIVES: Outer membrane vesicle (OMV) vaccines are used against outbreaks of capsular group B Neisseria meningitidis (MenB) caused by strains expressing particular PorA outer membrane proteins (OMPs). Ferric enterobactin receptor (FetA) is another variable OMP that induces type-specific bactericidal antibodies, and the combination of judiciously chosen PorA and FetA variants in vaccine formulations is a potential approach to broaden protection of such vaccines. METHODS: The OMV vaccine MenPF-1 was generated by genetically modifying N. meningitidis strain 44/76 to constitutively express FetA. Three doses of 25 µg or 50 µg of MenPF-1 were delivered intra-muscularly to 52 healthy adults. RESULTS: MenPF-1 was safe and well tolerated. Immunogenicity was measured by serum bactericidal assay (SBA) against wild-type and isogenic mutant strains. After 3 doses, the proportion of volunteers with SBA titres ≥1:4 (the putative protective titre) was 98% for the wild-type strain, and 77% for the strain 44/76 FetA(on)PorA(off) compared to 51% in the strain 44/76 FetA(off)PorA(off), demonstrating that vaccination with MenPF-1 simultaneously induced FetA and PorA bactericidal antibodies. CONCLUSION: This study provides a proof-of-concept for generating bactericidal antibodies against FetA after OMV vaccination in humans. Prevalence-based choice of PorA and FetA types can be used to formulate a vaccine for broad protection against MenB disease.

The nucleotide sequence and gene organization of the gerA spore germination operon of Bacillus subtilis 168.

The nucleotide sequence of the second and third genes in the Bacillus subtilis spore germination locus, gerA, has been determined and the amino acid (aa) sequence was derived. Two open reading frames (ORFs), corresponding to genes II and III, encode 364-aa residue and 373-aa residue polypeptides, respectively. The gene II product, Mr 41,257, would contain long stretches of hydrophobic aa residues and may be a membrane protein; the gene III product, Mr 42,363, is relatively hydrophilic but possesses an apparent signal peptide for transfer across, and perhaps localisation on, a membrane. The ORFs for genes I and II overlap by eleven codons and the termination codon of gene II overlaps the initiation codon of gene III. Insertional inactivation experiments using integrational plasmids have indicated that the gerA locus is a single transcriptional unit. The expression of the gerA genes has been studied using a lacZ transcriptional fusion; they constitute a developmentally regulated operon.

The nucleotide sequence of a spore germination gene (gerA) of Bacillus subtilis 168.

The nucleotide sequence of a 2.1-kb fragment of Bacillus subtilis DNA that contains part of the spore germination locus, gerA, has been determined. An open reading frame (ORF) of 1440 bp (480 codons) has been identified which corresponds to the previously located complementation unit I of the gerA locus. The orientations of transcription of the gerA and of the adjacent fumarase (citG) gene are divergent. The deduced polypeptide product of the gerA gene, of Mr 53 506, contains both hydrophobic and hydrophilic domains and is likely to be membrane-associated.

Prospects offered by genome studies for combating meningococcal disease by vaccination.

Meningococcal disease was first recognised and Neisseria meningitidis isolated as the causative agent over 100 years ago, but despite more than a century of research, attempts to eliminate this distressing illness have so far been thwarted. The main problem lies in the fact that N. meningitidis usually exists as a harmless commensal inhabitant of the human nasopharynx, the pathogenic state being the exception rather than the norm. As man is its only host, the meningococcus is uniquely adapted to this ecological niche and has evolved an array of mechanisms for evading clearance by the human immune response. Progress has been made in combating the disease by developing vaccines that target specific pathogenic serogroups of meningococci. However, a fully comprehensive vaccine that protects against all pathogenic strains is still just beyond reach. The publication of the genome sequences of two meningococcal strains, one each from serogroups A and B and the imminent completion of a third illustrates the extent of the problems to be overcome, namely the vast array of genetic mechanisms for the generation of meningococcal diversity. Fortunately, genome studies also provide new hope for solutions to these problems in the potential for a greater understanding of meningococcal pathogenesis and possibilities for the identification of new vaccine candidates. This review describes some of the approaches that are currently being used to exploit the information from meningococcal genome sequences and seeks to identify future prospects for combating meningococcal disease.

Peptide mimics elicit antibody responses against the outer-membrane lipooligosaccharide of group B neisseria meningitidis.

As an alternative approach towards the development of a meningococcal vaccine, the potential of peptide mimics of lipooligosaccharide (LOS) to elicit cross-reactive immune responses against LOS was investigated. The heptapeptides SMYGSYN and APARQLP were identified by enrichment from a coliphage display library with a LOS-specific monoclonal antibody. Mice immunised with these peptides conjugated to diphtheria toxoid elicited a total IgG response to LOS with geometric mean titres 2-4 times higher compared with non-immunised controls. There was an increase in LOS-specific IgG1 immunoglobulin, whereas specific IgG2a and IgG3 decreased slightly in response to immunisation. The data demonstrated that peptide mimics can elicit immune responses against meningococcal LOS.

Meningococcal vaccines and vaccine developments.

Despite rapid advances in the diagnosis of bacterial infections and the availability of effective antibiotics, meningococcal disease continues to represent a substantial public health problem for most countries (1-4). Disease usually develops rapidly, is notoriously difficult to distinguish from other febrile illnesses, and generally has a high case-fatality rate. The death of an otherwise fit and healthy individual can occur within a very short time from the first appearance of symptoms, those who survive frequently suffer from permanent tissue damage and neurological problems (4,5). Consequently, the development and implementation of effective immunoprophylaxis is a sine qua non for the comprehensive control of meningococcal disease. From an historical perspective, many meningococcal vaccines have been developed and evaluated in clinical trials; unfortunately, no vaccine so far offers comprehensive protection. This overview traces the development of the existing licensed vaccines and examines the prospects of vaccine candidates that are currently under development or subject to clinical evaluation.

Identification of Peptides that Mimic N. meningitidis LOS Epitopes Via the Use of Combinatorial Phage-Display Libraries.

Although capsular polysaccharide-based vaccines are effective at reducing the incidence of meningococcal disease caused by serogroups A, C, Y, and W135 (1-3), immunization against serogroup B disease using similar strategies has proven unsuccessful (4,5). The primary reason for this is that the α2,8-linked N-acetylneuraminic acid homopolymer expressed by serogroup B strains is poorly immunogenic in humans (6). Consequently, considerable effort has been devoted towards the development of alternative strategies for vaccination against serogroup B disease. Many of these newer strategies include the use of lipooligosaccharide (LOS) as a protective antigen (7). One of the approaches that we are currently pursuing involves the use of synthetic oligopeptides to stimulate antibody responses that are cross-reactive with LOS antigens expressed by serogroup B Neisseria meningitidis strains. An integral part of these studies has been the application of combinatorial phage-display technology. Described here is an overview of the methods that we have utilized to identify peptide mimics of LOS epitopes.

The immunogenicity of a conformationally restricted peptide mimetic of meningococcal lipooligosaccharide.

Life-threatening meningitis and septicaemia caused by Neisseria meningitidis are a public health priority, and their prevention by vaccination is a major objective. Meningococcal capsular polysaccharide-based vaccines are effective against the major invasive serogroups, except for serogroup B, the capsule of which mimics human polysaccharides and is poorly immunogenic. An alternative vaccine candidate that has the potential to offer cross-protection against antigenically diverse meningococci is the lipooligosaccharide (LOS). The structurally constrained peptide mimetic, C22, of a bactericidal antibody epitope within LOS was previously shown to elicit cross-reactive antibodies to meningococcal LOS when complexed to NeutrAvidintrade mark as a carrier protein. The immunogenicity of this antigen in H-2(d) (BALB/c) and H-2(k) (C3H/HeN) haplotype mice was further investigated. Anti-LOS immunoglobulin G (IgG) antibody titres increased with the vaccine dose and correlated with the anti-C22 peptide antibody titres in both haplotypes. Antigen-stimulated Th1/Th2 cytokine secretion by splenocytes and antibody isotypes indicated a Th2-type immune response with IgG1 antibodies and a low titre of IgG2b. There was no serum bactericidal activity observed against the meningococcus.

A gonococcal porA pseudogene: implications for understanding the evolution and pathogenicity of Neisseria gonorrhoeae.

Members of the genus Neisseria, including the human pathogens Neisseria meningitidis and Neisseria gonorrhoeae, express at least one member of a family of related porins. N. meningitidis is the only species known to express a second porin, the meningococcal serosubtyping antigen PorA, the most divergent member of this family. Unexpectedly, a porA gene was identified in the gonococcal genome. Both the gonococcal and meningococcal porA loci were adjacent to a homologue of the Escherichia coli greA gene, although the IS1106 element downstream of porA in some meningococci was absent in the gonococcus. Almost identical porA loci were present in four unrelated gonococcal isolates and clinical specimens from patients with gonorrhoea. Lack of PorA expression in the gonococcus resulted from mutations in the promoter region, which prevented transcription, and frameshift mutations in the coding region of the porA gene. Hybridization and amplification experiments, showing the absence of a porA gene in seven other Neisseria species, suggested that porA was acquired by a common ancestor of the gonococcus and meningococcus but inactivated in the gonococcus on speciation. This implies that, while advantageous during colonization of the upper respiratory tract, this protein has no function in, or hinders, colonization of the urogenital tract.

Serotype of Streptococcus pneumoniae capsular polysaccharide can modify the Th1/Th2 cytokine profile and IgG subclass response to pneumococal-CRM(197) conjugate vaccines in a murine model.

The cellular and antibody responses to type 14 and type 19F Streptococcus pneumoniae capsular polysaccharides (PS) conjugated to CRM(197) were investigated in a mouse model developed for pre-clinical evaluation and quality control of pneumococcal conjugate vaccines. Total IgG antibody and IgG subclasses against PS and the carrier protein for both conjugates were measured in addition to the T cell proliferation and cytokine profiles induced by these conjugates. While unconjugated PS 14 and 19F were at best only weakly immunogenic, both types of conjugate induced strong primary and secondary IgG responses to PS. The responses induced by the two conjugates to the carrier protein were very different; a high level of anti-CRM(197) IgG was induced only by the PS19F conjugate whereas a very weak response was induced by the PS14 conjugate. Interestingly, the IgG subclass distribution was different for the two conjugates; for PS19F conjugate, the IgG response was almost completely of IgG1 subclass with low levels of IgG3 and IgG2a while the response to PS14 conjugate was mainly of the IgG1 and IgG2a subclasses with a low level of IgG3. The anti-CRM(197) IgG subclass distribution was identical with that to the corresponding conjugated PS. Both types of conjugate induced strong T cell proliferation to recall antigens but induced different patterns of cytokine response in immune spleen cells which were indicative of a Th0 response or a mixture of Th1 and Th2 responses with a bias towards Th2 response in PS19F-CRM(197) immunised mice. In conclusion, PS14- and PS19F-CRM(197) conjugates induced different IgG subclass patterns as a result of inducing different patterns of cytokine response to the carrier protein. This indicates that the serotype of PS can modify the Th1/Th2 response to the carrier protein, which has a direct effect and can predict the IgG subclass of the PS response. Finally, we conclude that this model appears suitable for studying the immunogenicity and immune interaction of different components of multivalent pneumococcal conjugate vaccines and may be applicable to their pre-clinical evaluation and quality control.

Role of sigma H in expression of the fumarase gene (citG) in vegetative cells of Bacillus subtilis 168.

The fumarase gene (citG) of Bacillus subtilis is transcribed from two promoter regions, citGp1 and citGp2 (P1 and P2); the P2 promoter is used by the E sigma H form of RNA polymerase. In order to study the role of P1 and P2 in citG expression, the promoter region and various deletion derivatives that effectively separate P1 and P2 were fused to the Escherichia coli beta-galactosidase gene (lacZ) and introduced into the chromosome in single copy at the amyE locus. P1 functioned to provide a relatively low and stable basal level of fumarase activity throughout growth. In contrast, P2 activity was found to vary over at least a 50-fold range and was responsible for regulating fumarase activity during growth and sporulation in a rich medium and in response to changes in carbon source. To further investigate the role of sigma H in fumarase regulation, citGp2-lacZ fusions were introduced into a strain in which the expression of the chromosomal spoOH gene was under the control of the isopropylthiogalactopyranoside-inducible spac promoter. Induction of pspac did not lead to P2 induction, suggesting that citG expression is not regulated at the level of spoOH transcription.

The regulation of the fumarase (citG) gene of Bacillus subtilis 168.

The level of fumarase activity in Bacillus subtilis depends on the nutritional environment; in rich medium low vegetative levels increase towards the end of the exponential phase, whereas in minimal glucose medium levels are relatively high throughout growth. Analysis of the enzyme levels in spoO mutants has revealed that a functional spoOH gene is required for the efficient expression of fumarase in both media. This highlights a regulatory role for the spoOH gene product not only in control of postexponentially expressed genes, but also during vegetative growth in defined medium. S1 transcript mapping reveals three transcriptional startpoints for the fumarase structural gene (citG) in B. subtilis. The upstream promoter region P1, which appears to contain two transcriptional startpoints, is functional in both Escherichia coli and B. subtilis. Promoter P2, which is located closer to the structural gene, is only functional in B. subtilis. Transcription from this promoter is strictly dependent on a functional spoOH gene; this gene has recently been shown to encode a minor sigma factor.

Characterization of the fumarase gene of Bacillus subtilis 168 cloned and expressed in Escherichia coli K12.

The fumarase (citG) gene of Bacillus subtilis 168 has been identified in a collection of lambda phages carrying EcoRI-generated fragments of B. subtilis DNA. Regions of the cloned DNA have been subcloned into plasmid vectors, and the ability of prophages and multicopy plasmids to complement Escherichia coli and B. subtilis fumarate mutations has been examined. Two EcoRI fragments of 1.5 and 5.1 kb are both required for fumarase expression in E. coli and B. subtilis. The level of fumarase activity from a single copy of the B. subtilis citG gene expressed in E. coli is approximately one-tenth of that from the normal E. coli gene; the level is increased by expression from a pBR322-derived multicopy plasmid. The citG gene has been located within the cloned DNA by transposon mutagenesis and by expression studies, which have also identified a polypeptide of Mr 49000 as the product of the citG gene. The properties of a truncated derivative of this polypeptide have indicated the direction of transcription of the citG gene.

Identification of three complementation units in the gerA spore germination locus of Bacillus subtilis.

The gerA locus, mutations in which affect the germination response of spores to L-alanine and related amino acids, is contained within a 6-kilobase region of DNA cloned in phage and plasmid vectors. Fragments from this region, subcloned in the shuttle vector pHV33, were introduced into Bacillus subtilis, and their ability to complement chromosomal gerA mutations in a recE4 background was examined. Although the plasmids were somewhat unstable, it was possible to score complementation within spore-containing colonies on nutrient agar by their ability to reduce 2,3,5-triphenyltetrazolium chloride in an overlay. These studies have assigned the 10 gerA mutations tested to three complementation groups. An analysis of Tn1000 insertions into the cloned DNA of two relatively stable plasmids that together encompass the entire gerA region has identified more precisely the location and extent of the complementation units; recombination studies and in vitro mutagenesis were used to further delineate the extents of two of the units. The evidence suggests that the three complementation units are adjacent and that they are probably capable of separate transcription.

pIF21, a versatile donor of transposons Tn1, Tn5 and Tn7 for tagging and mobilizing cryptic and non-selectable plasmids.

A plasmid, pIF21, has been constructed that is able to donate transposons Tn1, Tn5 and Tn7. The transposons are located on a temperature-sensitive derivative of the incP1 plasmid pRP1, which is transferable to a wide range of Gram-negative genera.

Comparison of the class 1 outer membrane proteins of eight serological reference strains of Neisseria meningitidis.

Primers suitable for the amplification of the gene encoding the class 1 outer membrane protein of Neisseria meningitidis by the polymerase chain reaction (PCR) were designed from published DNA sequences and used to study the gene in eight meningococcal strains of different serogroup, serotype and subtype. At high annealing stringency one product, shown to correspond to the class 1 protein gene, was amplified from each strain. For three strains an additional smaller product, provisionally identified as the gene encoding the class 3 outer membrane protein, was amplified at lower annealing stringencies. Nucleotide sequence analysis of the PCR products corresponding to the class 1 proteins established the differences in the primary structure of the proteins between each of the subtypes and other outer-membrane proteins from Neisseria spp. These differences impose constraints on possible structural models of these proteins. Most amino acid sequence variation occurred in two domains of between 8 and 17 amino acids; there was an additional region which varied mainly between classes of outer membrane protein and there were nine conserved regions. Using appropriate primers it was possible to distinguish between class 1 outer membrane protein genes from strains of different subtypes by the PCR.