Impact of preexisting memory to seasonal A/H1N1 influenza virus on the immune response following vaccination against avian A/H5N1 virus.

Cross-protection against divergent strains of influenza virus is an objective of various vaccination approaches. B cells cross-neutralizing several influenza A heterosubtypes have been isolated from cultured human memory B cells (MBCs) and plasmablasts early after influenza vaccination or infection. However, a systematic assessment of the frequency of MBCs and plasmablasts in the blood of healthy individuals is lacking. Here, we show that under resting conditions about 45% of human adults never vaccinated nor exposed to avian A/H5N1 influenza have detectable circulating MBCs cross-reacting with H5N1. This proportion rises to 63.3% among subjects with a large pool of MBCs specific for seasonal H1N1 (i.e. frequency ≥1% of total IgG MBCs). Moreover, subjects with high baseline frequencies of H1N1-specific MBCs had an expansion of H5N1-specific MBCs producing H5-neutralizing antibodies already after the first dose of an MF59-adjuvanted H5N1 vaccine. These results suggest that H1N1-specific MBCs contain a subset of cells cross-reacting to H5. We propose that a proportion of human adults have a pool of H5/H1 cross-reactive MBCs that contribute to the rapid rise of the antibody response to divergent influenza strains. This may have implications on vaccination strategies aimed at counteracting future influenza pandemics.

Human heat shock protein (Hsp) 90 interferes with Neisseria meningitidis adhesin A (NadA)-mediated adhesion and invasion.

NadA (N eisseria meningitidisadhesin A), a meningococcal surface protein, mediates adhesion to and invasion of human cells, an activity in which host membrane proteins have been implicated. While investigating these host factors in human epithelial cells by affinity chromatography, we discovered an unanticipated interaction of NadA with heat shock protein (Hsp) 90, a molecular chaperone. The specific in vitro interaction of recombinant soluble NadA and Hsp90 was confirmed by co-immunoprecipitations, dot and far-Western blot. Intriguingly, ADP, but not ATP, was required for this association, and the Hsp90 inhibitor 17-AAG promoted complex formation. Hsp90 binding to an Escherichia coli strain used as carrier to express surface exposed NadA confirmed these results in live bacteria. We also examined RNA interference, plasmid-driven overexpression, addition of exogenous rHsp90 and 17-AAG inhibition in human epithelial cells to further elucidate the involvement of Hsp90 in NadA-mediated adhesion and invasion. Together, these data suggest an inverse correlation between the amount of host Hsp90 and the NadA adhesive/invasive phenotype. Confocal microscopy also demonstrated that meningococci interact with cellular Hsp90, a completely novel finding. Altogether our results show that variation of host Hsp90 expression or activity interferes with adhesive and invasive events driven by NadA.

Mapping of the Neisseria meningitidis NadA cell-binding site: relevance of predicted {alpha}-helices in the NH2-terminal and dimeric coiled-coil regions.

NadA is a trimeric autotransporter protein of Neisseria meningitidis belonging to the group of oligomeric coiled-coil adhesins. It is implicated in the colonization of the human upper respiratory tract by hypervirulent serogroup B N. meningitidis strains and is part of a multiantigen anti-serogroup B vaccine. Structure prediction indicates that NadA is made by a COOH-terminal membrane anchor (also necessary for autotranslocation to the bacterial surface), an intermediate elongated coiled-coil-rich stalk, and an NH(2)-terminal region involved in cell interaction. Electron microscopy analysis and structure prediction suggest that the apical region of NadA forms a compact and globular domain. Deletion studies proved that the NH(2)-terminal sequence (residues 24 to 87) is necessary for cell adhesion. In this study, to better define the NadA cell binding site, we exploited (i) a panel of NadA mutants lacking sequences along the coiled-coil stalk and (ii) several oligoclonal rabbit antibodies, and their relative Fab fragments, directed to linear epitopes distributed along the NadA ectodomain. We identified two critical regions for the NadA-cell receptor interaction with Chang cells: the NH(2) globular head domain and the NH(2) dimeric intrachain coiled-coil α-helices stemming from the stalk. This raises the importance of different modules within the predicted NadA structure. The identification of linear epitopes involved in receptor binding that are able to induce interfering antibodies reinforces the importance of NadA as a vaccine antigen.

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.

Human monocytes/macrophages are a target of Neisseria meningitidis Adhesin A (NadA).

Specific surface proteins of Neisseria meningitidis have been proposed to stimulate leukocytes during tissue invasion and septic shock. In this study, we demonstrate that the adhesin N. meningitidis Adhesin A (NadA) involved in the colonization of the respiratory epithelium by hypervirulent N. meningitidis B strains also binds to and activates human monocytes/macrophages. Expression of NadA on the surface on Escherichia coli does not increase bacterial-monocyte association, but a NadA-positive strain induced a significantly higher amount of TNF-alpha and IL-8 compared with the parental NadA-negative strain, suggesting that NadA has an intrinsic stimulatory action on these cells. Consistently, highly pure, soluble NadA(Delta351-405), a proposed component of an antimeningococcal vaccine, efficiently stimulates monocytes/macrophages to secrete a selected pattern of cytokines and chemotactic factors characterized by high levels of IL-8, IL-6, MCP-1, and MIP-1alpha and low levels of the main vasoactive mediators TNF-alpha and IL-1. NadA(Delta351-405) also inhibited monocyte apoptosis and determined its differentiation into a macrophage-like phenotype.

Monocyte-activation test to reliably measure the pyrogenic content of a vaccine: An in vitro pyrogen test to overcome in vivo limitations.

Pyrogen content is one of the critical quality attributes impacting the safety of a product, and there is an increasing need for assays that can reliably measure this attribute in vaccines. The Limulus amebocyte lysate (LAL) assay and the rabbit pyrogen test (RPT) are the canonical animal-based pyrogen tests currently used to release vaccines; however, there are several drawbacks associated with these tests when applied to Bexsero, intrinsically pyrogenic product, containing a meningococcal Outer Membrane Vesicle component. While the RPT, as applied to Bexsero at its given dilution, ensures safe vaccine, it is highly variable and prone to false positive results. On the other hand, the LAL assay although quantitative, can detect only endotoxin pyrogens and is not sufficient for monitoring the safety of Bexsero, which contains both LPS and non-endotoxin pyrogens. Being aware of these limitations of the RPT and LAL when applied to Bexsero, the Monocyte Activation Test (MAT) which is sensitive to both endotoxin and non-endotoxin based pyrogens has been developed as an alternative pyrogen test. Here, the development and the validation of a MAT assay adapted from the European pharmacopoeia for Bexsero, is described. The MAT assay is then used for monitoring the safety and consistency of Bexsero vaccines at release, providing great advantages in terms of reduced variability with respect to RPT, reduction of animal use, in line with the 3Rs principle concerning the protection of animals and faster time to market. In addition the correlation of the MAT to the RPT has been demonstrated supporting the replacement of the in vivo method and the potential application of the assay to other intrinsically pyrogenic vaccines.

Biotechnology and vaccines: application of functional genomics to Neisseria meningitidis and other bacterial pathogens.

Since its introduction, vaccinology has been very effective in preventing infectious diseases. However, in several cases, the conventional approach to identify protective antigens, based on biochemical, immunological and microbiological methods, has failed to deliver successful vaccine candidates against major bacterial pathogens. The recent development of powerful biotechnological tools applied to genome-based approaches has revolutionized vaccine development, biological research and clinical diagnostics. The availability of a genome provides an inclusive virtual catalogue of all the potential antigens from which it is possible to select the molecules that are likely to be more effective. Here, we describe the use of "reverse vaccinology", which has been successful in the identification of potential vaccines candidates against Neisseria meningitidis serogroup B and review the use of functional genomics approaches as DNA microarrays, proteomics and comparative genome analysis for the identification of virulence factors and novel vaccine candidates. In addition, we describe the potential of these powerful technologies in understanding the pathogenesis of various bacteria.

Neutralizing monoclonal antibodies to different clades of Influenza A H5N1 viruses.

Four IgG(1kappa) monoclonal antibodies (mAbs) against Influenza A/Chicken/Vietnam/8/2004 (H5N1) virus are described. Three of these showed neutralizing activities against H5N1 strains from clades 1, 2 and 3 using a retroviral pseudotype or live virus microneutralization assay. In the pseudotype assay, the IC(90) neutralizing titre range was >1600-51,200, and with the microneutralization was 80> or =10,240. MAb 1C1 showed strong neutralizing activities in both assays. All four mAbs reacted specifically to the immunogen by immunohistochemical staining and to A/Hong Kong/483/1997 (H5N1) and A/Thailand/1(KAN-1)/2004 (H5N1)-infected MDCK cells by immunofluorescence. ELISA titrations of the mAbs showed specificity for H5N1 haemagglutinin (HA) and no cross-reactivity to 15 other Influenza A subtypes. Only mAbs 1C1 and the non-neutralizing 1F7 reacted with HA(1), the cleaved subunit of HA, by Western blot. These results suggest that the mAbs recognize distinct or overlapping epitopes and will be useful reagents for construction of specific rapid point-of-care assays or for therapeutic use.

Human monoclonal antibodies in single chain fragment variable format with potent neutralization activity against influenza virus H5N1.

Effective diagnostic and therapeutic strategies are needed to control and combat the highly pathogenic avian influenza virus (AIV) subtype H5N1. To this end, we developed human monoclonal antibodies (mAbs) in single chain fragment variable (scFv) format towards the H5N1 avian influenza virus to gain new insights for the development of immunotherapy against human cases of H5N1. Using a biopanning based approach a large array of scFvs against H5N1 virus were isolated from the human semi-synthetic ETH-2 phage antibody library. H5N1 ELISA-positive scFvs with unique variable heavy (VH) and light (VL) chain gene sequences showed different biochemical properties and neutralization activity across H5N1 viral strains. In particular, the scFv clones AV.D1 and AV.C4 exerted a significant inhibition of the H5N1 A/Vietnam/1194/2004 virus infection in a pseudotype-based neutralization assay. Interestingly, these two scFvs displayed a cross-clade neutralizing activity versus A/whooping swan/Mongolia/244/2005 and A/Indonesia/5/2005 strains. These studies provide proof of the concept that human mAbs in scFv format with well-defined H5N1 recognition patterns and in vitro neutralizing activity can be easily and rapidly isolated by biopanning selection of an entirely artificial antibody repertoire using inactivated H5N1 virus as a bait.

Pseudoparticle neutralization is a reliable assay to measure immunity and cross-reactivity to H5N1 influenza viruses.

The standard serological methods present limitations for the measurement of immunity against H5N1 influenza strains. The hemagglutination inhibition (HI) assay lacks sensitivity and requires standardization, while the viral micro-neutralization (MN) assay needs handling of live virus. We produced pseudoparticles expressing hemagglutinin from clades 1 or 2 H5N1 in order to measure neutralizing antibodies in human sera after prime-boost vaccination with plain or MF59-adjuvanted H5N1 clade 1 subunit vaccines. Titers measured by pseudoparticle neutralization (PPN) assay significantly correlated with those measured by HI, single radial haemolysis or MN, with a PPN titer of 1:357 corresponding to an MN titer of 1:80. Notably, results from the PPN assay, confirm that MF59-H5N1 vaccine induces potent and long-lasting neutralizing antibody responses not only against the vaccine strain, but also against several heterologous clade 2 strains. Overall, the PPN assay represents a valid alternative to conventional serological methods for the evaluation of H5N1 vaccine immunogenicity.

Neisseria meningitidis NhhA is a multifunctional trimeric autotransporter adhesin.

NhhA, Neisseriahia/hsf homologue, or GNA0992, is an oligomeric outer membrane protein of Neisseria meningitidis, recently included in the family of trimeric autotransporter adhesins. In this study we present the structural and functional characterization of this protein. By expressing in Escherichia coli the full-length gene, deletion mutants and chimeric proteins of NhhA, we demonstrated that the last 72 C-terminal residues are able to allow trimerization and localization of the N-terminal protein domain to the bacterial surface. In addition, we investigated on the possible role of NhhA in bacterial-host interaction events. We assessed in vitro the ability of recombinant purified NhhA to bind human epithelial cells as well as laminin and heparan sulphate. Furthermore, we shown that E. coli strain expressing NhhA was able to adhere to epithelial cells, and observed a reduced adherence in a meningococcal isogenic MC58DeltaNhhA mutant. We concluded that this protein is a multifunctional adhesin, able to promote the bacterial adhesion to host cells and extracellular matrix components. Collectively, our results underline a putative role of NhhA in meningococcal pathogenesis and ascertain its structural and functional belonging to the emerging group of bacterial autotransporter adhesins with trimeric architecture.

A universal vaccine for serogroup B meningococcus.

Meningitis and sepsis caused by serogroup B meningococcus are two severe diseases that still cause significant mortality. To date there is no universal vaccine that prevents these diseases. In this work, five antigens discovered by reverse vaccinology were expressed in a form suitable for large-scale manufacturing and formulated with adjuvants suitable for human use. The vaccine adjuvanted by aluminum hydroxide induced bactericidal antibodies in mice against 78% of a panel of 85 meningococcal strains representative of the global population diversity. The strain coverage could be increased to 90% and above by the addition of CpG oligonucleotides or by using MF59 as adjuvant. The vaccine has the potential to conquer one of the most devastating diseases of childhood.

Neisseria meningitidis NadA is a new invasin which promotes bacterial adhesion to and penetration into human epithelial cells.

Neisseria meningitidis is a human pathogen, which is a major cause of sepsis and meningitis. The bacterium colonizes the upper respiratory tract of approximately 10% of humans where it lives as a commensal. On rare occasions, it crosses the epithelium and reaches the bloodstream causing sepsis. From the bloodstream it translocates the blood-brain barrier, causing meningitis. Although all strains have the potential to cause disease, a subset of them, which belongs to hypervirulent lineages, causes disease more frequently than others. Recently, we described NadA, a novel antigen of N. meningitidis, present in three of the four known hypervirulent lineages. Here we show that NadA is a novel bacterial invasin which, when expressed on the surface of Escherichia coli, promotes adhesion to and invasion into Chang epithelial cells. Deletion of the N-terminal globular domain of recombinant NadA or pronase treatment of human cells abrogated the adhesive phenotype. A hypervirulent strain of N. meningitidis where the nad A gene was inactivated had a reduced ability to adhere to and invade into epithelial cells in vitro. NadA is likely to improve the fitness of N. meningitidis contributing to the increased virulence of strains that belong to the hypervirulent lineages.

Two years into reverse vaccinology.

During the last century, several approaches have been used for the development of vaccines, going from the immunization with live-attenuated bacteria up to the formulation of the safer subunit vaccines. This conventional approach to vaccine development requires cultivation of the pathogen and its dissection using biochemical, immunological and microbiological methods. Although successful in several cases, this method is time-consuming and failed to provide a solution for many human pathogens. Now genomic approaches allow for the design of vaccines starting from the prediction of all antigens in silico, independently of their abundance and without the need to grow the microorganism in vitro. A new strategy, termed "Reverse Vaccinology", which has been successfully applied in the last few years, has revolutionized the approach to vaccine research. The Neisseria meningitidis serogroup B project, the first example of Reverse Vaccinology, as well as the application of this strategy to develop novel vaccines against other human pathogens are discussed.

The genome revolution in vaccine research.

The conventional approach to vaccine development is based on dissection of the pathogen using biochemical, immunological and microbiological methods. Although successful in several cases, this approach has failed to provide a solution to prevent several major bacterial infections. The availability of complete genome sequences in combination with novel advanced technologies, such as bioinformatics, microarrays and proteomics, have revolutionized the approach to vaccine development and provided a new impulse to microbial research. The genomic revolution allows the design of vaccines starting from the prediction of all antigens in silico, independently of their abundance and without the need to grow the pathogen in vitro. This new genome-based approach, which we have named "Reverse Vaccinology", has been successfully applied for Neisseria meningitidis serogroup B for which conventional strategies have failed to provide an efficacious vaccine. The concept of "Reverse Vaccinology" can be easily applied to all the pathogens for which vaccines are not yet available and can be extended to parasites and viruses.

NadA diversity and carriage in Neisseria meningitidis.

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.