Ala-Eddine Deghmane

Sequence Diversity of the Factor H Binding Protein Vaccine Candidate in Epidemiologically Relevant Strains of Serogroup B Neisseria meningitidis

BACKGROUND Recombinant forms of Neisseria meningitidis human factor H binding protein (fHBP) are undergoing clinical trials in candidate vaccines against invasive meningococcal serogroup B disease. We report an extensive survey and phylogenetic analysis of the diversity of fhbp genes and predicted protein sequences in invasive clinical isolates obtained in the period 2000-2006. METHODS Nucleotide sequences of fhbp genes were obtained from 1837 invasive N. meningitidis serogroup B (MnB) strains from the United States, Europe, New Zealand, and South Africa. Multilocus sequence typing (MLST) analysis was performed on a subset of the strains. RESULTS Every strain contained the fhbp gene. All sequences fell into 1 of 2 subfamilies (A or B), with 60%-75% amino acid identity between subfamilies and at least 83% identity within each subfamily. One fHBP sequence may have arisen via inter-subfamily recombination. Subfamily B sequences were found in 70% of the isolates, and subfamily A sequences were found in 30%. Multiple fHBP variants were detected in each of the common MLST clonal complexes. All major MLST complexes include strains in both subfamily A and subfamily B. CONCLUSIONS The diversity of strains observed underscores the importance of studying the distribution of the vaccine antigen itself rather than relying on common epidemiological surrogates such as MLST.

Down-regulation of pili and capsule of Neisseria meningitidis upon contact with epithelial cells is mediated by CrgA regulatory protein

The initial attachment of Neisseria meningitidis to the target cell surface appears to be largely pilus depend‐ent in capsulated bacteria. Intimate adhesion subsequently occurs to permit colonization. We recently reported that insertional inactivation of the crgA gene, which encodes a transcriptional regulator belonging to the LysR family, decreased meningococcal adhesion to epithelial cells and abolished intimate adhesion. In this report, we analyse expression of the pilE and sia genes, which are involved in the biosynthesis of pili and capsule respectively, during bacteria–host cell interactions. Western blotting, transcriptional fusion and reverse transcriptase polymerase chain reaction (RT‐PCR) analysis showed that the expression of these genes was downregulated during intimate adhesion. DNA‐binding assays, footprinting and RT‐PCR analysis indicated that this downregulation was directly mediated by the CrgA protein. The pilE and sia promoters were found to have a CrgA binding motif in common. These results strongly suggest that N. meningitidis displays an adaptive response upon cell contact. CrgA may play a central regulatory role in meningococcal adhesion, particularly in switching from initial to intimate adhesion by downregulating the bacterial surface structures that hinder this adhesion.

Correlations between Carbon Metabolism and Virulence in Bacteria

Bacteria have developed several mechanisms which allow the preferred utilization of the most efficiently metabolizable carbohydrates when these organisms are exposed to a mixture of carbon sources. Interestingly, the same or similar mechanisms are used by some pathogens to control various steps of their infection process. The efficient metabolism of a carbon source might serve as signal for proper fitness. Alternatively, the presence of a specific carbon source might indicate to bacterial cells that they thrive in infection-related organs, tissues or cells and that specific virulence genes should be turned on or switched off. Frequently, virulence gene regulators are affected by changes in carbon source availability. For example, expression of the gene encoding the Streptococcus pyogenes virulence regulator Mga is controlled by the classical carbon catabolite repression (CCR) mechanism operative in Firmicutes. The activity of PrfA, the major virulence regulator in Listeria monocytogenes, seems to be controlled by the phosphorylation state of phosphotransferase system(PTS) components. In Vibrio cholerae synthesis of HapR, which regulates the expression of genes required for motility, is controlled via the Crp/cAMP CCR mechanism, whereas synthesis of Salmonella enterica HilE, which represses genes in a pathogenicity island, is regulated by the carbohydrate-responsive, PTS-controlled Mlc.

Intimate adhesion of Neisseria meningitidis to human epithelial cells is under the control of the crgA gene, a novel LysR-type transcriptional regulator

PilC1, a pilus‐associated protein in Neisseria meningitidis, is a key element in initial meningococcal adhesion to target cells. A promoter element (CREN, contact regulatory element of Neisseria) is responsible for the transient induction of this gene upon cell contact. crgA (contact‐regulated gene A) encodes a transcriptional regulator whose expression is also induced upon cell contact from a promoter region similar to the CREN of pilC1. CrgA shows significant sequence homologies to LysR‐type transcriptional regulators. Its inactivation in meningococci provokes a dramatic reduction in bacterial adhesion to epithelial cells. Moreover, this mutant is unable to undergo intimate adhesion to epithelial cells or to provoke effacing of microvilli on infected cells. Purified CrgA is able to bind to pilC1 and crgA promoters, and CrgA seems to repress the expression of pilC1 and crgA. Our results support a dynamic model of bacteria–cell interaction involving a network of regulators acting in cascade. CrgA could be an intermediate regulator in such a network.

From tailor-made to ready-to-wear meningococcal B vaccines: longitudinal study of a clonal meningococcal B outbreak

BACKGROUND Outer-membrane-vesicle vaccines for meningococcal B outbreaks are complex and time consuming to develop. We studied the use of already available vaccine to control an outbreak caused by a genetically close strain. METHODS From 2006 to 2009, all individuals younger than 20 years living in the region of Normandy, France, in which an outbreak caused by a B:14:P1.7,16 strain occurred, were eligible to receive MenBvac, a Norwegian vaccine designed 20 years earlier against a strain sharing the same serosubtype (B:15:P1.7,16). The immunogenicity (in a randomly selected cohort of 400 children aged 1-5 years), safety, and epidemiological effect of the vaccination were assessed. FINDINGS 26,014 individuals were eligible to receive the vaccine. Shortage of vaccine production prompted start of the campaign in the highest incidence groups (1-5 years). 16,709 (64%) received a complete vaccination schedule of whom 13,589 (81%) received a 2+1 dose schedule (week 0, week 6, and month 8). At 6 weeks after the third dose, of 235 vaccinees for whom samples were available, 206 (88%) had a seroresponse, and 108 (56 %) of 193 had a seroresponse at 15 months. These results were similar to those described for tailor-made vaccines and their homologous strain. Only previously described adverse effects occurred. The incidence of B:14:P1.7,16 cases decreased significantly in the vaccine targeted population after the primary vaccination period (from 31·6 per 100,000 to 5·9 per 100,000; p=0·001). INTERPRETATION The ready-to-wear approach is reliable if epidemic and vaccine strains are genetically close. Other meningococcal B clonal outbreaks might benefit from this strategy; and previously described outer-membrane-vesicle vaccines can be effective against various strains. FUNDING French Ministry of Health.

Could the multicomponent meningococcal serogroup B vaccine (4CMenB) control Neisseria meningitidis capsular group X outbreaks in Africa

A new vaccine, 4CMenB, is composed of surface proteins of Neisseria meningitidis and is aimed to target serogroup B (MenB) isolates. The vaccine components are present in meningococcal isolates of other serogroups allowing potential use against meningococcal isolates belonging to non-B serogroups. Isolates of serogroup X (MenX) have been emerged in countries of the African meningitis belt. 4CMenB may offer a vaccine strategy against these isolates as there is no available capsule-based vaccine against MenX. We used the Meningococcal Antigen Typing System (MATS) to determine presence, diversity and levels of expression of 4CMenB antigens among 9 MenX isolates from several African countries in order to estimate the potential coverage of MenX by the 4CMenB vaccine. We performed bactericidal assays against these isolates, using pooled sera from 4CMenB-vaccinated infants, adolescents and adults. The African MenX isolates belonged to the same genotype but showed variation in the vaccine antigens. MATS data and bactericidal assays suggest coverage of the 9 African MenX isolates by 4CMenB but not of two unrelated MenX isolates from France. 4CMenB vaccine can be considered for further investigation to control MenX outbreaks in Africa.

The duality of virulence and transmissibility in Neisseria meningitidis

Neisseria meningitidis is a commensal bacterium of the human nasopharynx that occasionally provokes invasive disease. Carriage strains of N. meningitidis are heterogeneous, more frequent in nature and are transmitted among carriers. Disease is not a part of this transmission cycle and is caused by virulent strains. N. meningitidis is highly variable and variants that are modified in their virulence and/or transmissibility are continually generated. These events probably occur frequently, thus explaining not only the heterogeneous nature of meningococcal populations in carriers but probably also the evolutionary success of this human-restricted bacterium.

Hyperinvasive genotypes of Neisseria meningitidis in France

Clinical isolates of Neisseria meningitidis from cases of meningococcal disease, collected between January 2000 and December 2004, were identified and typed at the French National Reference Centre. A representative subset of 546 isolates from among 2882 isolates was further genotyped by multilocus sequence typing to determine their genetic lineages (clonal complexes) and the degree of diversification among different clonal complexes. Representative isolates of the main clonal complexes were tested for their virulence in mice and for proapoptotic effects on human epithelial cells. High genetic diversity in some genetic lineages (ST-32 and ST-41/44) was correlated with heterogeneity in virulence in mice and proapoptotic effects on human epithelial cells. In contrast, the homogeneous genetic structure of isolates of the ST-11 clonal complex, regardless of their serogroup, correlated positively with a fatal outcome of the infection, increased virulence in mice and increased proapoptotic effects on human epithelial cells.

Differential Modulation of TNF-α–Induced Apoptosis by Neisseria meningitidis

Infections by Neisseria meningitidis show duality between frequent asymptomatic carriage and occasional life-threatening disease. Bacterial and host factors involved in this balance are not fully understood. Cytopathic effects and cell damage may prelude to pathogenesis of isolates belonging to hyper-invasive lineages. We aimed to analyze cell–bacteria interactions using both pathogenic and carriage meningococcal isolates. Several pathogenic isolates of the ST-11 clonal complex and carriage isolates were used to infect human epithelial cells. Cytopathic effect was determined and apoptosis was scored using several methods (FITC-Annexin V staining followed by FACS analysis, caspase assays and DNA fragmentation). Only pathogenic isolates were able to induce apoptosis in human epithelial cells, mainly by lipooligosaccharide (endotoxin). Bioactive TNF-α is only detected when cells were infected by pathogenic isolates. At the opposite, carriage isolates seem to provoke shedding of the TNF-α receptor I (TNF-RI) from the surface that protect cells from apoptosis by chelating TNF-α. Ability to induce apoptosis and inflammation may represent major traits in the pathogenesis of N. meningitidis. However, our data strongly suggest that carriage isolates of meningococci reduce inflammatory response and apoptosis induction, resulting in the protection of their ecological niche at the human nasopharynx.

Emergence of New Virulent Neisseria meningitidis Serogroup C Sequence Type 11 Isolates in France

In France, there have been variations in the incidence of invasive meningococcal infection due to serogroup C isolates. Infection peaks were observed in 1992 and 2003 that involved isolates of phenotypes C:2a:P1.5,2 and/or C:2a:P1.5, which belong to the sequence type 11 (ST-11) clonal complex. We report an emergence of isolates belonging to the ST-11 clonal complex since 2003. These isolates displayed a new phenotype, C:2a:P1.7,1, caused infections that occurred as clusters, and were associated with increased infection severity and high virulence in mice. These isolates may be responsible for a peak in the incidence of serogroup C meningococcal infection in France, for which there is no routine vaccination to date.