Selective and non-selective bottlenecks as drivers of the evolution of hypermutable bacterial loci.

Bottlenecks reduce the size of the gene pool within populations of all life forms with implications for their subsequent survival. Here, we examine the effects of bottlenecks on bacterial commensal-pathogens during transmission between, and dissemination within, hosts. By reducing genetic diversity, bottlenecks may alter individual or population-wide adaptive potential. A diverse range of hypermutable mechanisms have evolved in infectious agents that allow for rapid generation of genetic diversity in specific genomic loci as opposed to the variability arising from increased genome-wide mutation rates. These localised hypermutable mechanisms include multi-gene phase variation (PV) of outer membrane components, multi-allele PV of restriction systems and recombination-driven antigenic variation. We review selected experimental and theoretical (mathematical) models pertaining to the hypothesis that localised hypermutation (LH) compensates for fitness losses caused by bottlenecks and discuss whether bottlenecks have driven the evolution of hypermutable loci.

Genomic Surveillance of 4CMenB Vaccine Antigenic Variants among Disease-Causing Neisseria meningitidis Isolates, United Kingdom, 2010-2016.

In September 2015, 4CMenB meningococcal vaccine was introduced into the United Kingdom infant immunization program without phase 3 trial information. Understanding the effect of this program requires enhanced surveillance of invasive meningococcal disease (IMD) Neisseria meningitidis isolates and comparison with prevaccination isolates. Bexsero Antigen Sequence Types (BASTs) were used to analyze whole-genome sequences of 3,073 prevaccine IMD N. meningitidis isolates obtained during 2010-2016. Isolates exhibited 803 BASTs among 31 clonal complexes. Frequencies of antigen peptide variants were factor H binding protein 1, 13.4%; Neisserial heparin-binding antigen 2, 13.8%; Neisseria adhesin A 8, 0.8%; and Porin A-VR2:P1.4,10.9%. In 2015-16, serogroup B isolates showed the highest proportion (35.7%) of exact matches to >1 Bexsero components. Serogroup W isolates showed the highest proportion (93.9%) of putatively cross-reactive variants of Bexsero antigens. Results highlighted the likely role of cross-reactive antigens. BAST surveillance of meningococcal whole-genome sequence data is rapid, scalable, and portable and enables international comparisons of isolates.

Genome sequencing of disease and carriage isolates of nontypeable Haemophilus influenzae identifies discrete population structure.

One of the main hurdles for the development of an effective and broadly protective vaccine against nonencapsulated isolates of Haemophilus influenzae (NTHi) lies in the genetic diversity of the species, which renders extremely difficult the identification of cross-protective candidate antigens. To assess whether a population structure of NTHi could be defined, we performed genome sequencing of a collection of diverse clinical isolates representative of both carriage and disease and of the diversity of the natural population. Analysis of the distribution of polymorphic sites in the core genome and of the composition of the accessory genome defined distinct evolutionary clades and supported a predominantly clonal evolution of NTHi, with the majority of genetic information transmitted vertically within lineages. A correlation between the population structure and the presence of selected surface-associated proteins and lipooligosaccharide structure, known to contribute to virulence, was found. This high-resolution, genome-based population structure of NTHi provides the foundation to obtain a better understanding, of NTHi adaptation to the host as well as its commensal and virulence behavior, that could facilitate intervention strategies against disease caused by this important human pathogen.

Crystal Structure of a Complex of Surfactant Protein D (SP-D) and Haemophilus influenzae Lipopolysaccharide Reveals Shielding of Core Structures in SP-D-Resistant Strains.

The carbohydrate recognition domains (CRDs) of lung collectin surfactant protein D (SP-D) recognize sugar patterns on the surface of lung pathogens and promote phagocytosis. Using Haemophilus influenzae Eagan strains expressing well-characterized lipopolysaccharide (LPS) surface structures of various levels of complexity, we show that bacterial recognition and binding by SP-D is inversely related to LPS chain extent and complexity. The crystal structure of a biologically active recombinant trimeric SP-D CRD complexed with a delipidated Eagan 4A LPS suggests that efficient LPS recognition by SP-D requires multiple binding interactions utilizing the three major ligand-binding determinants in the SP-D binding pocket, with Ca-dependent binding of inner-core heptose accompanied by interaction of anhydro-Kdo (4,7-anhydro-3-deoxy-d-manno-oct-2-ulosonic acid) with Arg343 and Asp325. Combined with enzyme-linked immunosorbent assays (ELISAs) and fluorescence-activated cell sorter (FACS) binding analyses, our results show that extended LPS structures previously thought to be targets for collectins are important in shielding the more vulnerable sites in the LPS core, revealing a mechanism by which pathogens with complex LPS extensions efficiently evade a first-line mucosal innate immune defense. The structure also reveals for the first time the dominant form of anhydro-Kdo.

In-silico prediction and deep-DNA sequencing validation indicate phase variation in 115 Neisseria meningitidis genes.

BACKGROUND: The Neisseria meningitidis (Nm) chromosome shows a high abundance of simple sequence DNA repeats (SSRs) that undergo stochastic, reversible mutations at high frequency. This mechanism is reflected in an extensive phenotypic diversity that facilitates Nm adaptation to dynamic environmental changes. To date, phase-variable phenotypes mediated by SSRs variation have been experimentally confirmed for 26 Nm genes. RESULTS: Here we present a population-scale comparative genomic analysis that identified 277 genes and classified them into 52 strong, 60 moderate and 165 weak candidates for phase variation. Deep-coverage DNA sequencing of single colonies grown overnight under non-selective conditions confirmed the presence of high-frequency, stochastic variation in 115 of them, providing circumstantial evidence for their phase variability. We confirmed previous observations of a predominance of variable SSRs within genes for components located on the cell surface or DNA metabolism. However, in addition we identified an unexpectedly broad spectrum of other metabolic functions, and most of the variable SSRs were predicted to induce phenotypic changes by modulating gene expression at a transcriptional level or by producing different protein isoforms rather than mediating on/off translational switching through frameshifts. Investigation of the evolutionary history of SSR contingency loci revealed that these loci were inherited from a Nm ancestor, evolved independently within Nm, or were acquired by Nm through lateral DNA exchange. CONCLUSIONS: Overall, our results have identified a broader and qualitatively different phenotypic diversification of SSRs-mediated stochastic variation than previously documented, including its impact on central Nm metabolism.

The role of host and microbial factors in the pathogenesis of pneumococcal bacteraemia arising from a single bacterial cell bottleneck.

The pathogenesis of bacteraemia after challenge with one million pneumococci of three isogenic variants was investigated. Sequential analyses of blood samples indicated that most episodes of bacteraemia were monoclonal events providing compelling evidence for a single bacterial cell bottleneck at the origin of invasive disease. With respect to host determinants, results identified novel properties of splenic macrophages and a role for neutrophils in early clearance of pneumococci. Concerning microbial factors, whole genome sequencing provided genetic evidence for the clonal origin of the bacteraemia and identified SNPs in distinct sub-units of F0/F1 ATPase in the majority of the ex vivo isolates. When compared to parental organisms of the inoculum, ex-vivo pneumococci with mutant alleles of the F0/F1 ATPase had acquired the capacity to grow at low pH at the cost of the capacity to grow at high pH. Although founded by a single cell, the genotypes of pneumococci in septicaemic mice indicate strong selective pressure for fitness, emphasising the within-host complexity of the pathogenesis of invasive disease.

Neisseria meningitidis is structured in clades associated with restriction modification systems that modulate homologous recombination.

Molecular data on a limited number of chromosomal loci have shown that the population of Neisseria meningitidis (Nm), a deadly human pathogen, is structured in distinct lineages. Given that the Nm population undergoes substantial recombination, the mechanisms resulting in the evolution of these lineages, their persistence in time, and the implications for the pathogenicity of the bacterium are not yet completely understood. Based on whole-genome sequencing, we show that Nm is structured in phylogenetic clades. Through acquisition of specific genes and through insertions and rearrangements, each clade has acquired and remodeled specific genomic tracts, with the potential to impact on the commensal and virulence behavior of Nm. Despite this clear evidence of a structured population, we confirm high rates of detectable recombination throughout the whole Nm chromosome. However, gene conversion events were found to be longer within clades than between clades, suggesting a DNA cleavage mechanism associated with the phylogeny of the species. We identify 22 restriction modification systems, probably acquired by horizontal gene transfer from outside of the species/genus, whose distribution in the different strains coincides with the phylogenetic clade structure. We provide evidence that these clade-associated restriction modification systems generate a differential barrier to DNA exchange consistent with the observed population structure. These findings have general implications for the emergence of lineage structure and virulence in recombining bacterial populations, and they could provide an evolutionary framework for the population biology of a number of other bacterial species that show contradictory population structure and dynamics.

Qualitative and quantitative assessment of meningococcal antigens to evaluate the potential strain coverage of protein-based vaccines.

A unique multicomponent vaccine against serogroup B meningococci incorporates the novel genome-derived proteins fHbp, NHBA, and NadA that may vary in sequence and level of expression. Measuring the effectiveness of such vaccines, using the accepted correlate of protection against invasive meningococcal disease, could require performing the serum bactericidal assay (SBA) against many diverse strains for each geographic region. This approach is impractical, especially for infants, where serum volumes are very limited. To address this, we developed the meningococcal antigen typing system (MATS) by combining a unique vaccine antigen-specific ELISA, which detects qualitative and quantitative differences in antigens, with PorA genotyping information. The ELISA correlates with killing of strains by SBA and measures both immunologic cross-reactivity and quantity of the antigens NHBA, NadA, and fHbp. We found that strains exceeding a threshold value in the ELISA for any of the three vaccine antigens had ≥80% probability of being killed by immune serum in the SBA. Strains positive for two or more antigens had a 96% probability of being killed. Inclusion of multiple different antigens in the vaccine improves breadth of coverage and prevents loss of coverage if one antigen mutates or is lost. The finding that a simple and high-throughput assay correlates with bactericidal activity is a milestone in meningococcal vaccine development. This assay allows typing of large panels of strains and prediction of coverage of protein-based meningococcal vaccines. Similar assays may be used for protein-based vaccines against other bacteria.

Inactivation of Haemophilus influenzae lipopolysaccharide biosynthesis genes interferes with outer membrane localization of the hap autotransporter.

Nontypeable Haemophilus influenzae is a major cause of localized respiratory tract disease and initiates infection by colonizing the nasopharynx. Colonization requires adherence to host epithelial cells, which is mediated by surface proteins such as the Hap adhesin. In this study, we identified a relationship between Hap levels in the outer membrane and lipopolysaccharide (LPS) biosynthesis enzymes. We found that mutation of the rfaF, pgmB, lgtC, kfiC, orfE, rfbP, lsgB, or lsgD genes, which are involved in the synthesis of the LPS oligosaccharide core in H. influenzae strain Rd/HapS243A, resulted in loss of Hap in the bacterial outer membrane and a decrease in hap transcript levels. In contrast, the same mutations had no effect on outer membrane localization of H. influenzae P5 or IgA1 protease or levels of p5 or iga1 transcripts, suggesting a Hap-specific effect. Elimination of the HtrA periplasmic protease resulted in a return of Hap to the outer membrane and restoration of hap transcript levels. Consistently, in lgtC phase-off bacteria, Hap was absent from the outer membrane, and hap transcript levels were reduced. Hap localization and hap transcript levels were not related to LPS size but to the functions of the LPS biosynthesis enzymes themselves. We speculate that the lack of certain LPS biosynthesis enzymes causes Hap to mislocalize and accumulate in the periplasm, where it is degraded by HtrA. This degradation then leads to a decrease in hap transcript levels. Together, these data highlight a novel interplay between Hap and LPS biosynthesis that can influence H. influenzae interactions with the host.

The next decade of vaccines: societal and scientific challenges.

Vaccines against microbial diseases have improved the health of millions of people. In the next decade and beyond, many conceptual and technological scientific advances offer extraordinary opportunities to expand the portfolio of immunisations against viral and bacterial diseases and to pioneer the first vaccines against human parasitic and fungal diseases. Scientists in the public and private sectors are motivated as never before to bring about these innovations in immunisation. Many societal factors threaten to compromise realisation of the public health gains that immunisation can achieve in the next decade and beyond--understanding these factors is imperative. Vaccines are typically given to healthy individuals and safety issues loom high on the list of public concerns. The public needs to regain confidence in immunisation and trust the organisations responsible for the research, development, and implementation of vaccines. In the past, by use of a judicious amalgam of knowledge and empiricism, successful vaccines were largely developed by microbiologists who identified antigens that induced immune responses to conserved pathogen components. In the future, vaccines need to be developed against deadly diseases for which this strategy is often not feasible because of the extensive antigenic variability of relevant pathogens. High microbial diversity means that immunity after natural infection is often ineffective for prevention of disease on subsequent exposure, for example in HIV infection and malaria. Additionally, vaccines need to be generated to protect the people who are most vulnerable because of age or underlying diseases. Thus, in the future, a much deeper understanding of the immunological challenges--including the diversifying role of host genetics and environmental factors, leading perhaps to more personalised approaches-will be the touchstone for rational design and development of adjuvants that result in novel safe and effective vaccines.

Investigations into genome diversity of Haemophilus influenzae using whole genome sequencing of clinical isolates and laboratory transformants.

BACKGROUND: Haemophilus influenzae is an important human commensal pathogen associated with significant levels of disease. High-throughput DNA sequencing was used to investigate differences in genome content within this species. RESULTS: Genomic DNA sequence was obtained from 85 strains of H. influenzae and from other related species, selected based on geographical site of isolation, disease association and documented genotypic and phenotypic differences. When compared by Mauve alignment these indicated groupings of H. influenzae that were consistent with previously published analyses; capsule expressing strains fell into two distinct groups and those of serotype b (Hib) were found in two closely positioned lineages. For 18 Hib strains representing both lineages we found many discrete regions (up to 40% of the total genome) displaying sequence variation when compared to a common reference strain. Evidence that this naturally occurring pattern of inter-strain variation in H. influenzae can be mediated by transformation was obtained through sequencing DNA obtained from a pool of 200 independent transformants of a recipient (strain Rd) using donor DNA from a heterologous Hib strain (Eagan). CONCLUSION: Much of the inter-strain variation in genome sequence in H. influenzae is likely the result of inter-strain exchanges of DNA, most plausibly through transformation.

Diurnal Differences in Intracellular Replication Within Splenic Macrophages Correlates With the Outcome of Pneumococcal Infection.

Circadian rhythms affect the progression and severity of bacterial infections including those caused by Streptococcus pneumoniae, but the mechanisms responsible for this phenomenon remain largely elusive. Following advances in our understanding of the role of replication of S. pneumoniae within splenic macrophages, we sought to investigate whether events within the spleen correlate with differential outcomes of invasive pneumococcal infection. Utilising murine invasive pneumococcal disease (IPD) models, here we report that infection during the murine active phase (zeitgeber time 15; 15h after start of light cycle, 3h after start of dark cycle) resulted in significantly faster onset of septicaemia compared to rest phase (zeitgeber time 3; 3h after start of light cycle) infection. This correlated with significantly higher pneumococcal burden within the spleen of active phase-infected mice at early time points compared to rest phase-infected mice. Whole-section confocal microscopy analysis of these spleens revealed that the number of pneumococci is significantly higher exclusively within marginal zone metallophilic macrophages (MMMs) known to allow intracellular pneumococcal replication as a prerequisite step to the onset of septicaemia. Pneumococcal clusters within MMMs were more abundant and increased in size over time in active phase-infected mice compared to those in rest phase-infected mice which decreased in size and were present in a lower percentage of MMMs. This phenomenon preceded significantly higher levels of bacteraemia alongside serum IL-6 and TNF-α concentrations in active phase-infected mice following re-seeding of pneumococci into the blood. These data greatly advance our fundamental knowledge of pneumococcal infection by linking susceptibility to invasive pneumococcal infection to variation in the propensity of MMMs to allow persistence and replication of phagocytosed bacteria. These findings also outline a somewhat rare scenario whereby the active phase of an organism's circadian cycle plays a seemingly counterproductive role in the control of invasive infection.

The macrophage scavenger receptor A is host-protective in experimental meningococcal septicaemia.

Macrophage Scavenger Receptor A (SR-A) is a major non-opsonic receptor for Neisseria meningitidis on mononuclear phagocytes in vitro, and the surface proteins NMB0278, NMB0667, and NMB1220 have been identified as ligands for SR-A. In this study we ascertain the in vivo role of SR-A in the recognition of N. meningitidis MC58 (serogroup B) in a murine model of meningococcal septicaemia. We infected wild-type and SR-A(-/-) animals intraperitoneally with N. meningitidis MC58 and monitored their health over a period of 50 hours. We also determined the levels of bacteraemia in the blood and spleen, and measured levels of the pro-inflammatory cytokine interleukin-6 (IL-6). The health of SR-A(-/-) animals deteriorated more rapidly, and they showed a 33% reduction in survival compared to wild-type animals. SR-A(-/-) animals consistently exhibited higher levels of bacteraemia and increased levels of IL-6, compared to wild-type animals. Subsequently, we constructed a bacterial mutant (MC58-278-1220) lacking two of the SR-A ligands, NMB0278 and NMB1220. Mutation of NMB0667 proved to be lethal. When mice were infected with the mutant bacteria MC58-278-1220, no significant differences could be observed in the health, survival, bacteraemia, and cytokine production between wild-type and SR-A(-/-) animals. Overall, mutant bacteria appeared to cause less severe symptoms of septicaemia, and a competitive index assay showed that higher levels of wild-type bacteria were recovered when animals were infected with a 1ratio1 ratio of wild-type MC58 and mutant MC58-278-1220 bacteria. These data represent the first report of the protective role of SR-A, a macrophage-restricted, non-opsonic receptor, in meningococcal septicaemia in vivo, and the importance of the recognition of bacterial protein ligands, rather than lipopolysaccharide.

Darwin, microbes and evolution by natural selection.

Born 200 years ago, Darwin's revolutionary ideas were derived largely from his observations on life forms that evolved relatively recently, including various flowering plants, worms, birds and domesticated animals. Yet, life appeared on planet earth close to 4 billion years ago in the form of unicellular organisms collectively called bacteria. It was only shortly after "On the Origin of Species" was published (1859) that the "germ theory" of infectious diseases was formulated. Microbes (viruses, bacteria, fungi and microparasites) received scant mention in Darwin's writings, although pioneers of the Golden Age of Bacteriology, such as Louis Pasteur (1822-1895), were contemporaries. Today, microbes offer extraordinary testimony and powerful model systems of direct relevance to the essentials of Darwinian selection, such as understanding microbial-host interactions, the evolution of pathogens and the emergence of drug- or vaccine-related resistance.

Interaction of Klebsiella pneumoniae with tissue macrophages in a mouse infection model and ex-vivo pig organ perfusions: an exploratory investigation.

BACKGROUND: Hypervirulent Klebsiella pneumoniae (hvKp) strains of capsule type K1 and K2 cause invasive infections associated with hepatic abscesses, which can be difficult to treat and are frequently associated with relapsing infections. Other K pneumoniae strains (non-hvKp), including lineages that have acquired carbapenem resistance, do not manifest this pathology. In this work we aimed to test the hypothesis that within-macrophage replication is a key mechanism underpinning abscess formation in hvKp infections. METHODS: In this exploratory investigation, to study the pathophysiology of abscess formation, mice were intravenously infected with 106 colony forming units (CFU) of either hvKp isolates (six strains) or non-hvKp isolates (seven strains). Intracellular bacterial replication and neutrophil influx in liver and spleen was quantified by fluorescence microscopy of sliced cryopreserved organs of mice collected 30 min, 6 h, and 24 h after infection with the aim to provide data of bacterial association to Kupffer cells in the liver and to the different tissue macrophages in the spleen. Microbiological and microscopy analysis of an ex-vivo model of pig liver and spleen infection were used to confirm within-macrophage replication. Pig organs were perfused with heparinised, autologous pig's blood and injected with 6·5 × 107 CFU of hvKp K2 sequence type 25 strain GMR151. Blood and tissue biopsies collected before infection and 30 min, 1 h, 2 h, 3 h, 4 h, and 5 h after infection were used to measure bacterial counts and to identify the subcellular localisation of bacteria by immunohistochemistry analysis. FINDINGS: We show that hvKp resisted phagocyte-mediated clearance and replicated in mouse liver macrophages to form clusters 6 h after infection, with a mean of 7·0 bacteria per Kupffer cell (SD 6·2); however, non-hvKp were efficiently cleared (mean 1·5 bacteria per cell [SD 1·1]). HvKp infection promoted neutrophil recruitment to sites of infection, which in the liver resulted in histopathological signs of abscess formation as early as 24 h post-infection. Experiments in pig organs which share a high functional and anatomical resemblance to human organs, provided strong evidence for the propensity of hvKp to replicate within the hepatic macrophages. INTERPRETATION: These findings show subversion of innate immune processes in the liver by K pneumoniae and resistance to Kupffer cell mediated clearance as an explanation for the propensity of hvKp strains to cause hepatic abscesses. FUNDING: University of Oxford and a Royal Society Wolfson grant funded biosafety facility.

Splenic macrophages as the source of bacteraemia during pneumococcal pneumonia.

BACKGROUND: Severe community-acquired pneumococcal pneumonia is commonly associated with bacteraemia. Although it is assumed that the bacteraemia solely derives from pneumococci entering the blood from the lungs it is unknown if other organs are important in the pathogenesis of bacteraemia. Using three models, we tested the relevance of the spleen in pneumonia-associated bacteraemia. METHODS: We used human spleens perfused ex vivo to explore permissiveness to bacterial replication, a non-human primate model to check for splenic involvement during pneumonia and a mouse pneumonia-bacteraemia model to demonstrate that splenic involvement correlates with invasive disease. FINDINGS: Here we present evidence that the spleen is the reservoir of bacteraemia during pneumonia. We found that in the human spleen infected with pneumococci, clusters with increasing number of bacteria were detectable within macrophages. These clusters also were detected in non-human primates. When intranasally infected mice were treated with a non-therapeutic dose of azithromycin, which had no effect on pneumonia but concentrated inside splenic macrophages, bacteria were absent from the spleen and blood and importantly mice had no signs of disease. INTERPRETATION: We conclude that the bacterial load in the spleen, and not lung, correlates with the occurrence of bacteraemia. This supports the hypothesis that the spleen, and not the lungs, is the major source of bacteria during systemic infection associated with pneumococcal pneumonia; a finding that provides a mechanistic basis for using combination therapies including macrolides in the treatment of severe community-acquired pneumococcal pneumonia. FUNDING: Oxford University, Wolfson Foundation, MRC, NIH, NIHR, and MRC and BBSRC studentships supported the work.

Investigating the candidacy of LPS-based glycoconjugates to prevent invasive meningococcal disease: immunology of glycoconjugates with high carbohydrate loading.

We investigated the immune responses of rabbits that were immunised with lipopolysaccharide (LPS)-based glycoconjugates by measuring the reactivity of the derived sera to a panel of selected wild-type and mutant strains of Neisseria meningitidis. In all cases, high titers of antibodies capable of recognising LPS elaborating the identical structure as presented on the immunising glycoconjugate were obtained, and in most cases the derived sera also recognised heterologous strains including wild-type, but at lower titers. However, although serum bactericidal antibodies were consistently obtained against strains elaborating the same LPS structure as the immunising antigen, this functional response was not observed against wild-type strains. We identified several potentially competing neo-epitopes that had been introduced via our conjugation strategies, which might compete with the conserved inner core oligosaccharide target region, thus reducing the antibody titers to epitopes which could facilitate bactericidal killing. This study has therefore identified key factors that are crucial to control in order to increase the likelihood of obtaining bactericidal antibodies to wild-type meningococcal cells with LPS-derived glycoconjugates. Glycoconjugates utilised in this study, have been found to contain epitopes that do not contribute to the derivation of antibodies that may facilitate bactericidal killing of wild-type strains and must be avoided in future LPS-based glycoconjugate preparations.

Haemophilus influenzae biofilms: hypothesis or fact?

Many publications state that nontypeable Haemophilus influenzae (NTHi) produces biofilms. Here, we review many of the publications that have led to acceptance by some that NTHi expresses a biofilm-specific phenotype as a distinct part of its life cycle. Biofilm formation was originally invoked to explain the failure to culture NTHi from middle-ear effusions, recalcitrance to antibiotics and its pathogenic behaviour. We argue that the current evidence for NTHi biofilm formation in vitro and in vivo is inconclusive. We consider that NTHi biofilm is hypothesis not fact, and although it might yet prove to be correct, there has been little or no consideration of alternative interpretations for the in vitro and in vivo observations. Uncritical acceptance of a distinctive NTHi biofilm phenotype has the potential to mislead and could confuse and compromise research efforts aimed at improving management and prevention of NTHi diseases of the human respiratory tract.

The Development of a Vaccine Against Meningococcus B Using Reverse Vaccinology.

The discovery of vaccine antigens through whole genome sequencing (WGS) contrasts with the classical hypothesis-driven laboratory-based analysis of microbes to identify components to elicit protective immunity. This radical change in scientific direction and action in vaccine research is captured in the term reverse vaccinology. The complete genome sequence of an isolate of Neisseria meningitidis serogroup B (MenB) was systematically analyzed to identify proteins predicted to be secreted or exported to the outer membrane. This identified hundreds of genes coding for potential surface-exposed antigens. These were amplified, cloned in expression vectors and used to immunize mice. Antisera against 350 recombinant antigens were obtained and analyzed in a panel of immunological assays from which 28 were selected as potentially protective based on the -antibody dependent, complement mediated- serum bactericidal activity assay. Testing of these candidate vaccine antigens, using a large globally representative strain collection of Neisseria species isolated from cases of disease and carriage, indicated that no single component would be sufficient to induce broad coverage and that a "universal" vaccine should contain multiple antigens. The final choice of antigens to be included was based on cross-protective ability, assayed by serum bactericidal activity and maximum coverage of the extensive antigenic variability of MenB strains. The resulting multivalent vaccine formulation selected consisted of three recombinant antigens (Neisserial Heparin Binding Antigen or NHBA, Factor H binding protein or fHbp and Neisseria Adhesin A or NadA). To improve immunogenicity and potential strain coverage, an outer membrane vesicle component obtained from the epidemic New Zealand strain (OMVNz) was added to the formulation to create a four component vaccine, called 4CMenB. A series of phase 2 and 3 clinical trials were conducted to evaluate safety and tolerability and to estimate the vaccine effectiveness of human immune responses at different ages and how these were affected by various factors including concomitant vaccine use and lot-to-lot consistency. 4CMenB was approved in Europe in 2013 and introduced in the National Immunization Program in the UK starting from September 2015 when the vaccine was offered to all newborns using a 2, 4, and 12 months schedule., The effectiveness against invasive MenB disease measured at 11 months after the study start and 5 months after the second vaccination was 83% and there have been no safety concerns.

Structural analysis of the lipopolysaccharide from nontypeable Haemophilus influenzae strain R2846.

We here report the lipopolysaccharide (LPS) structures expressed by nontypeable Haemophilus influenzae R2846, a strain whose complete genome sequence has recently been obtained. Results were obtained by using NMR techniques and ESI-MS on O-deacylated LPS and core oligosaccharide material (OS) as well as ESI-MS (n) on permethylated dephosphorylated OS. A beta- d-Glc p-(1-->4)- d-alpha- d-Hep p-(1-->6)-beta- d-Glc p-(1-->4) unit was found linked to the proximal heptose (HepI) of the conserved triheptosyl inner-core moiety, l-alpha- d-Hep p-(1-->2)-[ PEtn-->6]- l-alpha- d-Hep p-(1-->3)- l-alpha- d-Hep p-(1-->5)-[ PPEtn-->4]-alpha-Kdo-(2-->6)-lipid A. The beta- d-Glc p (GlcI) linked to HepI was also branched with oligosaccharide extensions from O-4 and O-6. O-4 of GlcI was substituted with sialyllacto- N-neotetraose [alpha-Neu5Ac-(2-->3)-beta- d-Gal p-(1-->4)-beta- d-Glc pNAc-(1-->3)-beta- d-Gal p-(1-->4)-beta- d-Glc p-(1-->] and the related structure [( PEtn-->6)-alpha- d-Gal pNAc-(1-->6)-beta- d-Gal p-(1-->4)-beta- d-Glc pNAc-(1-->3)-beta- d-Gal p-(1-->4)-beta- d-Glc p-(1-->]. The distal heptose (HepIII) was substituted at O-2 by beta- d-Gal. Phosphate, phosphoethanolamine, phosphocholine, acetate, and glycine were found to substitute the core oligosaccharide. Two heptosyltransferase genes, losB1 and losB2, have been identified from the R2846 genome sequence and are candidates to add the noncore heptose to the LPS. Mutant strain R2846 losB1 did not show dd-heptose in the extension from HepI but still contained minor quantities of ld-heptose at the same position, indicating that the losB1 gene is required to add dd-heptose to GlcI. The LPS from strain R2846 losB1/ losB2 expressed no noncore heptose, consistent with losB2 directing the addition of ld-heptose.