Stefania Bambini

Vaccination against Neisseria meningitidis Using Three Variants of the Lipoprotein GNA1870

Sepsis and meningitis caused by serogroup B meningococcus are devastating diseases of infants and young adults, which cannot yet be prevented by vaccination. By genome mining, we discovered GNA1870, a new surface-exposed lipoprotein of Neisseria meningitidis that induces high levels of bactericidal antibodies. The antigen is expressed by all strains of N. meningitidis tested. Sequencing of the gene in 71 strains representative of the genetic and geographic diversity of the N. meningitidis population, showed that the protein can be divided into three variants. Conservation within each variant ranges between 91.6 to 100%, while between the variants the conservation can be as low as 62.8%. The level of expression varies between strains, which can be classified as high, intermediate, and low expressors. Antibodies against a recombinant form of the protein elicit complement-mediated killing of the strains that carry the same variant and induce passive protection in the infant rat model. Bactericidal titers are highest against those strains expressing high yields of the protein; however, even the very low expressors are efficiently killed. The novel antigen is a top candidate for the development of a new vaccine against meningococcus.

NadA, a Novel Vaccine Candidate of Neisseria meningitidis

Neisseria meningitidis is a human pathogen, which, in spite of antibiotic therapy, is still a major cause of mortality due to sepsis and meningitis. Here we describe NadA, a novel surface antigen of N. meningitidis that is present in 52 out of 53 strains of hypervirulent lineages electrophoretic types (ET) ET37, ET5, and cluster A4. The gene is absent in the hypervirulent lineage III, in N. gonorrhoeae and in the commensal species N. lactamica and N. cinerea. The guanine/cytosine content, lower than the chromosome, suggests acquisition by horizontal gene transfer and subsequent limited evolution to generate three well-conserved alleles. NadA has a predicted molecular structure strikingly similar to a novel class of adhesins (YadA and UspA2), forms high molecular weight oligomers, and binds to epithelial cells in vitro supporting the hypothesis that NadA is important for host cell interaction. NadA induces strong bactericidal antibodies and is protective in the infant rat model suggesting that this protein may represent a novel antigen for a vaccine able to control meningococcal disease caused by three hypervirulent lineages.

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.

Predicted strain coverage of a meningococcal multicomponent vaccine (4CMenB) in Europe: a qualitative and quantitative assessment.

BACKGROUND A novel multicomponent vaccine against meningococcal capsular group B (MenB) disease contains four major components: factor-H-binding protein, neisserial heparin binding antigen, neisserial adhesin A, and outer-membrane vesicles derived from the strain NZ98/254. Because the public health effect of the vaccine, 4CMenB (Novartis Vaccines and Diagnostics, Siena, Italy), is unclear, we assessed the predicted strain coverage in Europe. METHODS We assessed invasive MenB strains isolated mainly in the most recent full epidemiological year in England and Wales, France, Germany, Italy, and Norway. Meningococcal antigen typing system (MATS) results were linked to multilocus sequence typing and antigen sequence data. To investigate whether generalisation of coverage applied to the rest of Europe, we also assessed isolates from the Czech Republic and Spain. FINDINGS 1052 strains collected from July, 2007, to June, 2008, were assessed from England and Wales, France, Germany, Italy, and Norway. All MenB strains contained at least one gene encoding a major antigen in the vaccine. MATS predicted that 78% of all MenB strains would be killed by postvaccination sera (95% CI 63-90, range of point estimates 73-87% in individual country panels). Half of all strains and 64% of covered strains could be targeted by bactericidal antibodies against more than one vaccine antigen. Results for the 108 isolates from the Czech Republic and 300 from Spain were consistent with those for the other countries. INTERPRETATION MATS analysis showed that a multicomponent vaccine could protect against a substantial proportion of invasive MenB strains isolated in Europe. Monitoring of antigen expression, however, will be needed in the future. FUNDING Novartis Vaccines and Diagnostics.

Distribution and genetic variability of three vaccine components in a panel of strains representative of the diversity of serogroup B meningococcus

With the aim of studying the molecular diversity of the antigens of a new recombinant vaccine against meningococcus serogroup B, the three genes coding for the main vaccine components GNA (Genome-derived Neisseria Antigen) 1870 (fHbp, factor H Binding Protein), GNA1994 (NadA, Neisseria adhesin A) and GNA2132 were sequenced in a panel of 85 strains collected worldwide and selected as representative of the serogroup B meningococcal diversity. No correlations were found between vaccine antigen variability and serogroup, geographic area and year of isolation. Although a relevant clustering was found with MLST clonal complexes, each showing an almost specific antigen variant repertoire, the prediction of the antigen assortment was not possible on the basis of MLST alone. Therefore, classification of meningococcus on the basis of MLST only is not sufficient to predict vaccine antigens diversity. Sequencing each gene in the different strains will be important to evaluate antigen conservation and assortment and to allow a future prediction of potential vaccine coverage.

NadA Diversity and Carriage in Neisseria meningitidis

ABSTRACT NadA is a novel vaccine candidate recently identified in Neisseria meningitidis and involved in adhesion to host tissues. The nadA gene has been found in approximately 50% of the strains isolated from patients and in three of the four hypervirulent lineages of non-serogroup A strains. Here we investigated the presence of the nadA gene in 154 meningococcal strains isolated from healthy people (carrier strains). Only 25 (16.2%) of the 154 carrier isolates harbored the nadA gene. The commensal species Neisseria lactamica was also found not to have the nadA gene. Eighteen of the carrier strains belonged to the ET-5 and ET-37 hypervirulent clusters, indicating that only the 5.1% of the genuine carrier population actually harbored nadA (7 of 136 strains). Five of the seven strains harbored a novel allele of the nadA gene that was designated nadA4. The NadA4 protein was present on the bacterial surface as heat-stable high-molecular-weight oligomers. Antibodies against the recombinant NadA4 protein were bactericidal against homologous strains, whereas the activity against other NadA alleles was weak. In conclusion, the nadA gene segregates differently in the population of strains isolated from healthy individuals and in the population of strains isolated from patients. The presence of NadA can therefore be used as a tool to study the dynamics of meningococcal infections and understand why this bacterium, which is mostly a commensal, can become a severe pathogen.

Characterization of fHbp, nhba (gna2132), nadA, porA, and sequence type in group B meningococcal case isolates collected in England and Wales during January 2008 and potential coverage of an investigational group B meningococcal vaccine.

ABSTRACT Invasive disease caused by meningococcal capsular groups A, C, W-135, and Y is now preventable by means of glycoconjugate vaccines that target their respective polysaccharide capsules. The capsule of group B meningococci (MenB) is poorly immunogenic and may induce autoimmunity. Vaccines based on the major immunodominant surface porin, PorA, are effective against clonal epidemics but, thus far, have a limited scope of coverage against the wider MenB population at large. In an alternative approach, the first-generation, investigational, recombinant MenB (rMenB) plus outer membrane vesicle (OMV) (rMenB-OMV) vaccine contains a number of relatively conserved surface proteins, fHBP, NHBA (previously GNA2132), and NadA, alongside PorA P1.4-containing OMVs from the New Zealand MeNZB vaccine. MenB currently accounts for approximately 90% of cases of meningococcal disease in England and Wales. To assess potential rMenB-OMV vaccine coverage of pathogenic MenB isolates within this region, all English and Welsh MenB case isolates from January 2008 (n = 87) were genetically characterized with respect to fHBP, NHBA, NadA, and PorA. Alleles for fHbp, nhba, and porA were identified in all of the isolates, of which 22% were also found to harbor nadA alleles. On the basis of genotypic data and predicted immunological cross-reactivity, the potential level of rMenB-OMV vaccine coverage in England and Wales ranges from 66% to 100%.

Prevalence and genetic diversity of candidate vaccine antigens among invasive Neisseria meningitidis isolates in the United States

Neisseria meningitidis (Nm) serogroups B, C and Y are the major causes of meningococcal diseases in the United States. NmB accounts for ∼1/3 of the disease but no licensed vaccine is yet available. Two candidate vaccines are being developed specifically to target NmB, but may also provide protection against other serogroups. To assess the potential impact of these vaccines on NmB and other serogroups causing disease in the US, we determined the prevalence, genetic diversity and epidemiological characteristics of three candidate antigen genes in Nm isolates collected through Active Bacterial Core surveillance (ABCs), a population-based active surveillance program. fHbp was detected in all NmB, NmY and NmW135 isolates. Eleven NmC isolates contain fHbp with a single base-pair deletion creating a frame shift in the C-terminal region. Among NmB, 59% were FHbp subfamily/variant B/v1 and 41% A/v2-3. Among NmC and NmY, 39% and 3% were B/v1, respectively. nadA was detected in 39% of NmB, 61% of NmC and 4% of NmY. Among isolates tested, nhbA was present in all NmB and 96% of non-B. For the subset of strains sequenced for NadA and NhbA, pairwise identity was greater than 93% and 78%, respectively. The proportion of FHbp subfamily/variant was different between ABCs site and year, but no linear temporal trend was observed. Although assessment of the vaccine coverage also requires understanding of the antigen expression and the ability to induce bactericidal activity, our finding that all isolates contain one or more antigen genes suggests these candidate vaccines may protect against multiple Nm serogroups.

The use of genomics in microbial vaccine development.

Vaccination is one of the most effective tools for the prevention of infectious diseases. The availability of complete genome sequences, together with the progression of high-throughput technologies such as functional and structural genomics, has led to a new paradigm in vaccine development. Pan-genomic reverse vaccinology, with the comparison of sequence data from multiple isolates of the same species of a pathogen, increases the opportunity of the identification of novel vaccine candidates. Overall, the conventional empiric approach to vaccine development is being replaced by vaccine design. The recent development of synthetic genomics may provide a further opportunity to design vaccines.

Characterization of fHbp, nhba (gna2132), nadA, porA, sequence type (ST), and genomic presence of IS1301 in group B meningococcal ST269 clonal complex isolates from England and Wales.

ABSTRACT Highly effective glycoconjugate vaccines exist against four of the five major pathogenic groups of meningococci: A, C, W-135, and Y. An equivalent vaccine against group B meningococci (menB) has remained elusive due to the poorly immunogenic capsular polysaccharide. A promising alternative, the investigational recombinant menB (rMenB)- outer membrane vesicle (OMV) vaccine, contains fHBP, NHBA (previously GNA2132), NadA, and outer membrane vesicles (OMVs) from the New Zealand MeNZB vaccine. MenB currently accounts for 90% of meningococcal disease in England and Wales, where the multilocus sequence type (ST) 269 (ST269) clonal complex (cc269) has recently expanded to account for a third of menB cases. To assess the potential cc269 coverage of the rMenB-OMV vaccine, English and Welsh cc269 isolates from the past decade were genetically characterized with respect to fHBP, NHBA, and NadA. All of the isolates harbored fHbp and nhba alleles, while 98% of the cc269 isolates were devoid of nadA. Subvariant profiling of fHbp, nhba, and porA against STs revealed the presence of two broadly distinct and well-defined clusters of isolates, centered around ST269 and ST275, respectively. An additional molecular marker, insertion sequence IS1301, was found to be present in 100% and <2% of isolates of the respective clusters. On the basis of the genetic data, the potential rMenB-OMV coverage of cc269 in England and Wales is high (up to 100%) within both clusters. Expression studies and serum bactericidal antibody assays will serve to enhance predictions of coverage and will augment ongoing studies regarding the significance of IS1301 within the ST269 cluster.