Publications by authors named "Davide Serruto"

45 Publications

High coverage of diverse invasive meningococcal serogroup B strains by the 4-component vaccine 4CMenB in Australia, 2007-2011: Concordant predictions between MATS and genetic MATS.

Hum Vaccin Immunother 2021 Apr 13:1-9. Epub 2021 Apr 13.

Queensland Paediatric Infectious Disease Laboratory, Children's Health Queensland Hospitals and Health Service, Queensland Children's Hospital, Brisbane, Australia.

Meningococcal serogroup B (MenB) accounts for an important proportion of invasive meningococcal disease (IMD). The 4-component vaccine against MenB (4CMenB) is composed of factor H binding protein (fHbp), neisserial heparin-binding antigen (NHBA), adhesin A (NadA), and outer membrane vesicles of the New Zealand strain with Porin 1.4. A meningococcal antigen typing system (MATS) and a fully genomic approach, genetic MATS (gMATS), were developed to predict coverage of MenB strains by 4CMenB. We characterized 520 MenB invasive disease isolates collected over a 5-year period (January 2007-December 2011) from all Australian states/territories by multilocus sequence typing and estimated strain coverage by 4CMenB. The clonal complexes most frequently identified were ST-41/44 CC/Lineage 3 (39.4%) and ST-32 CC/ET-5 CC (23.7%). The overall MATS predicted coverage was 74.6% (95% coverage interval: 61.1%-85.6%). The overall gMATS prediction was 81.0% (lower-upper limit: 75.0-86.9%), showing 91.5% accuracy compared with MATS. Overall, 23.7% and 13.1% (MATS) and 26.0% and 14.0% (gMATS) of isolates were covered by at least 2 and 3 vaccine antigens, respectively, with fHbp and NHBA contributing the most to coverage. When stratified by year of isolate collection, state/territory and age group, MATS and gMATS strain coverage predictions were consistent across all strata. The high coverage predicted by MATS and gMATS indicates that 4CMenB vaccination may have an impact on the burden of MenB-caused IMD in Australia. gMATS can be used in the future to monitor variations in 4CMenB strain coverage over time and geographical areas even for non-culture confirmed IMD cases.
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http://dx.doi.org/10.1080/21645515.2021.1904758DOI Listing
April 2021

The role of vaccines in combatting antimicrobial resistance.

Nat Rev Microbiol 2021 May 4;19(5):287-302. Epub 2021 Feb 4.

GSK, Siena, Italy.

The use of antibiotics has enabled the successful treatment of bacterial infections, saving the lives and improving the health of many patients worldwide. However, the emergence and spread of antimicrobial resistance (AMR) has been highlighted as a global threat by different health organizations, and pathogens resistant to antimicrobials cause substantial morbidity and death. As resistance to multiple drugs increases, novel and effective therapies as well as prevention strategies are needed. In this Review, we discuss evidence that vaccines can have a major role in fighting AMR. Vaccines are used prophylactically, decreasing the number of infectious disease cases, and thus antibiotic use and the emergence and spread of AMR. We also describe the current state of development of vaccines against resistant bacterial pathogens that cause a substantial disease burden both in high-income countries and in low- and medium-income countries, discuss possible obstacles that hinder progress in vaccine development and speculate on the impact of next-generation vaccines against bacterial infectious diseases on AMR.
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http://dx.doi.org/10.1038/s41579-020-00506-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7861009PMC
May 2021

Self-Assembling Nanoparticles Usher in a New Era of Vaccine Design.

Cell 2019 03;176(6):1245-1247

GSK, 53100 Siena, Italy.

In this issue, Marcandalli et al. (2019) report a self-assembling nanoparticle bearing an antigen from respiratory syncytial virus. This is the first time the structure, stability, and adjuvanticity of an antigen have been rationally designed at the atomic level and incorporated in one vaccine.
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http://dx.doi.org/10.1016/j.cell.2019.02.008DOI Listing
March 2019

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

Vaccine 2019 06 14;37(29):3754-3760. Epub 2018 Nov 14.

GSK srl, Via Fiorentina 1, 53100 Siena, Italy. Electronic address:

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.
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http://dx.doi.org/10.1016/j.vaccine.2018.10.082DOI Listing
June 2019

Neisserial Heparin Binding Antigen (NHBA) Contributes to the Adhesion of Neisseria meningitidis to Human Epithelial Cells.

PLoS One 2016 25;11(10):e0162878. Epub 2016 Oct 25.

GSK Vaccines, Siena, Italy.

Neisserial Heparin Binding Antigen (NHBA) is a surface-exposed lipoprotein ubiquitously expressed by Neisseria meningitidis strains and an antigen of the Bexsero® vaccine. NHBA binds heparin through a conserved Arg-rich region that is the target of two proteases, the meningococcal NalP and human lactoferrin (hLf). In this work, in vitro studies showed that recombinant NHBA protein was able to bind epithelial cells and mutations of the Arg-rich tract abrogated this binding. All N-terminal and C-terminal fragments generated by NalP or hLf cleavage, regardless of the presence or absence of the Arg-rich region, did not bind to cells, indicating that a correct positioning of the Arg-rich region within the full length protein is crucial. Moreover, binding was abolished when cells were treated with heparinase III, suggesting that this interaction is mediated by heparan sulfate proteoglycans (HSPGs). N. meningitidis nhba knockout strains showed a significant reduction in adhesion to epithelial cells with respect to isogenic wild-type strains and adhesion of the wild-type strain was inhibited by anti-NHBA antibodies in a dose-dependent manner. Overall, the results demonstrate that NHBA contributes to meningococcal adhesion to epithelial cells through binding to HSPGs and suggest a possible role of anti-Bexsero® antibodies in the prevention of colonization.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0162878PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5079597PMC
June 2017

Global transcriptome analysis reveals small RNAs affecting Neisseria meningitidis bacteremia.

PLoS One 2015 7;10(5):e0126325. Epub 2015 May 7.

Novartis Vaccines and Diagnotics, Siena, Italy.

Most bacterial small RNAs (sRNAs) are post-transcriptional regulators involved in adaptive responses, controlling gene expression by modulating translation or stability of their target mRNAs often in concert with the RNA chaperone Hfq. Neisseria meningitides, the leading cause of bacterial meningitis, is able to adapt to different host niches during human infection. However, only a few sRNAs and their functions have been fully described to date. Recently, transcriptional expression profiling of N. meningitides in human blood ex vivo revealed 91 differentially expressed putative sRNAs. Here we expanded this analysis by performing a global transcriptome study after exposure of N. meningitides to physiologically relevant stress signals (e.g. heat shock, oxidative stress, iron and carbon source limitation). and we identified putative sRNAs that were differentially expressed in vitro. A set of 98 putative sRNAs was obtained by analyzing transcriptome data and 8 new sRNAs were validated, both by Northern blot and by primer extension techniques. Deletion of selected sRNAs caused attenuation of N. meningitides infection in the in vivo infant rat model, leading to the identification of the first sRNAs influencing meningococcal bacteremia. Further analysis indicated that one of the sRNAs affecting bacteremia responded to carbon source availability through repression by a GntR-like transcriptional regulator. Both the sRNA and the GntR-like regulator are implicated in the control of gene expression from a common network involved in energy metabolism.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0126325PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4423775PMC
April 2016

Staphylococcal Esx proteins modulate apoptosis and release of intracellular Staphylococcus aureus during infection in epithelial cells.

Infect Immun 2014 Oct 21;82(10):4144-53. Epub 2014 Jul 21.

Novartis Vaccines and Diagnostics, Siena, Italy Division of Microbiology and Infection, Warwick Medical School, University of Warwick, Coventry, United Kingdom

The opportunistic pathogen Staphylococcus aureus is one of the major causes of health care-associated infections. S. aureus is primarily an extracellular pathogen, but it was recently reported to invade and replicate in several host cell types. The ability of S. aureus to persist within cells has been implicated in resistance to antimicrobials and recurrent infections. However, few staphylococcal proteins that mediate intracellular survival have been identified. Here we examine if EsxA and EsxB, substrates of the ESAT-6-like secretion system (Ess), are important during intracellular S. aureus infection. The Esx proteins are required for staphylococcal virulence, but their functions during infection are unclear. While isogenic S. aureus esxA and esxB mutants were not defective for epithelial cell invasion in vitro, a significant increase in early/late apoptosis was observed in esxA mutant-infected cells compared to wild-type-infected cells. Impeding secretion of EsxA by deleting C-terminal residues of the protein also resulted in a significant increase of epithelial cell apoptosis. Furthermore, cells transfected with esxA showed an increased protection from apoptotic cell death. A double mutant lacking both EsxA and EsxB also induced increased apoptosis but, remarkably, was unable to escape from cells as efficiently as the single mutants or the wild type. Thus, using in vitro models of intracellular staphylococcal infection, we demonstrate that EsxA interferes with host cell apoptotic pathways and, together with EsxB, mediates the release of S. aureus from the host cell.
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http://dx.doi.org/10.1128/IAI.01576-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4187876PMC
October 2014

Neisseria meningitidis NalP cleaves human complement C3, facilitating degradation of C3b and survival in human serum.

Proc Natl Acad Sci U S A 2014 Jan 23;111(1):427-32. Epub 2013 Dec 23.

Research Center, Novartis Vaccines and Diagnostics, 53100 Siena, Italy.

The complement system is a crucial component of the innate immune response against invading bacterial pathogens. The human pathogen Neisseria meningitidis (Nm) is known to possess several mechanisms to evade the complement system, including binding to complement inhibitors. In this study, we describe an additional mechanism used by Nm to evade the complement system and survive in human blood. Using an isogenic NalP deletion mutant and NalP complementing strains, we show that the autotransporter protease NalP cleaves C3, the central component of the complement cascade. The cleavage occurs 4 aa upstream from the natural C3 cleavage site and produces shorter C3a-like and longer C3b-like fragments. The C3b-like fragment is degraded in the presence of the complement regulators (factors H and I), and this degradation results in lower deposition of C3b on the bacterial surface. We conclude that NalP is an important factor to increase the survival of Nm in human serum.
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http://dx.doi.org/10.1073/pnas.1321556111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3890809PMC
January 2014

Identification of a novel zinc metalloprotease through a global analysis of Clostridium difficile extracellular proteins.

PLoS One 2013 26;8(11):e81306. Epub 2013 Nov 26.

Novartis Vaccines and Diagnostics, Siena, Italy.

Clostridium difficile is a major cause of infectious diarrhea worldwide. Although the cell surface proteins are recognized to be important in clostridial pathogenesis, biological functions of only a few are known. Also, apart from the toxins, proteins exported by C. difficile into the extracellular milieu have been poorly studied. In order to identify novel extracellular factors of C. difficile, we analyzed bacterial culture supernatants prepared from clinical isolates, 630 and R20291, using liquid chromatography-tandem mass spectrometry. The majority of the proteins identified were non-canonical extracellular proteins. These could be largely classified into proteins associated to the cell wall (including CWPs and extracellular hydrolases), transporters and flagellar proteins. Seven unknown hypothetical proteins were also identified. One of these proteins, CD630_28300, shared sequence similarity with the anthrax lethal factor, a known zinc metallopeptidase. We demonstrated that CD630_28300 (named Zmp1) binds zinc and is able to cleave fibronectin and fibrinogen in vitro in a zinc-dependent manner. Using site-directed mutagenesis, we identified residues important in zinc binding and enzymatic activity. Furthermore, we demonstrated that Zmp1 destabilizes the fibronectin network produced by human fibroblasts. Thus, by analyzing the exoproteome of C. difficile, we identified a novel extracellular metalloprotease that may be important in key steps of clostridial pathogenesis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0081306PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3841139PMC
October 2014

Functional genomics studies of the human pathogen Neisseria meningitidis.

Brief Funct Genomics 2013 Jul 30;12(4):328-40. Epub 2013 May 30.

Novartis Vaccines & Diagnostics, Siena, Italy.

Neisseria meningitidis is a strictly human pathogen and is one of the major causes of septicemia and meningitis worldwide. Functional genomics approaches have been extensively applied to study how N. meningitidis adapts to grow and survive in different human niches encountered during the infection. DNA microarrays performed in in vitro and ex vivo conditions have revealed changes in the transcriptome profiles of N. meningitidis upon interaction with human cells and after incubation in human serum and blood. Mutagenesis studies allowed detecting mutants in genes crucial for N. meningitidis colonization and systemic infection. The analysis of N. meningitidis genomes has been also successful in the identification of vaccine candidates used to develop an effective protein-based vaccine. The application of all these approaches revealed the potential to identify new virulence factors and vaccine candidates and to assign functions to previously uncharacterized genes providing new insights in the biology and pathogenesis of N. meningitidis.
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http://dx.doi.org/10.1093/bfgp/elt018DOI Listing
July 2013

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

Lancet Infect Dis 2013 May 13;13(5):416-25. Epub 2013 Feb 13.

University of Würzburg, Institute for Hygiene and Microbiology, Würzburg, Germany.

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.
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http://dx.doi.org/10.1016/S1473-3099(13)70006-9DOI Listing
May 2013

Multiple factors modulate biofilm formation by the anaerobic pathogen Clostridium difficile.

J Bacteriol 2013 Feb 21;195(3):545-55. Epub 2012 Nov 21.

Novartis Vaccines and Diagnostics, Siena, Italy.

Bacteria within biofilms are protected from multiple stresses, including immune responses and antimicrobial agents. The biofilm-forming ability of bacterial pathogens has been associated with increased antibiotic resistance and chronic recurrent infections. Although biofilms have been well studied for several gut pathogens, little is known about biofilm formation by anaerobic gut species. The obligate anaerobe Clostridium difficile causes C. difficile infection (CDI), a major health care-associated problem primarily due to the high incidence of recurring infections. C. difficile colonizes the gut when the normal intestinal microflora is disrupted by antimicrobial agents; however, the factors or processes involved in gut colonization during infection remain unclear. We demonstrate that clinical C. difficile strains, i.e., strain 630 and the hypervirulent strain R20291, form structured biofilms in vitro, with R20291 accumulating substantially more biofilm. Microscopic and biochemical analyses show multiple layers of bacteria encased in a biofilm matrix containing proteins, DNA, and polysaccharide. Employing isogenic mutants, we show that virulence-associated proteins, Cwp84, flagella, and a putative quorum-sensing regulator, LuxS, are all required for maximal biofilm formation by C. difficile. Interestingly, a mutant in Spo0A, a transcription factor that controls spore formation, was defective for biofilm formation, indicating a possible link between sporulation and biofilm formation. Furthermore, we demonstrate that bacteria in clostridial biofilms are more resistant to high concentrations of vancomycin, a drug commonly used for treatment of CDI. Our data suggest that biofilm formation by C. difficile is a complex multifactorial process and may be a crucial mechanism for clostridial persistence in the host.
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http://dx.doi.org/10.1128/JB.01980-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3554014PMC
February 2013

Analysis of the regulated transcriptome of Neisseria meningitidis in human blood using a tiling array.

J Bacteriol 2012 Nov 14;194(22):6217-32. Epub 2012 Sep 14.

Novartis Vaccines and Diagnostics, Siena, Italy.

Neisseria meningitidis is the major cause of septicemia and meningococcal meningitis. During the course of infection, the bacterium must adapt to different host environments as a crucial factor for survival and dissemination; in particular, one of the crucial factors in N. meningitidis pathogenesis is the ability to grow and survive in human blood. We recently showed that N. meningitidis alters the expression of 30% of the open reading frames (ORFs) of the genome during incubation in human whole blood and suggested the presence of fine regulation at the gene expression level in order to control this step of pathogenesis. In this work, we used a customized tiling oligonucleotide microarray to define the changes in the whole transcriptional profile of N. meningitidis in a time course experiment of ex vivo bacteremia by incubating bacteria in human whole blood and then recovering RNA at different time points. The application of a newly developed bioinformatic tool to the tiling array data set allowed the identification of new transcripts--small intergenic RNAs, cis-encoded antisense RNAs, mRNAs with extended 5' and 3' untranslated regions (UTRs), and operons--differentially expressed in human blood. Here, we report a panel of expressed small RNAs, some of which can potentially regulate genes involved in bacterial metabolism, and we show, for the first time in N. meningitidis, extensive antisense transcription activity. This analysis suggests the presence of a circuit of regulatory RNA elements used by N. meningitidis to adapt to proliferate in human blood that is worthy of further investigation.
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http://dx.doi.org/10.1128/JB.01055-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3486417PMC
November 2012

Streptococcus pyogenes SpyCEP influences host-pathogen interactions during infection in a murine air pouch model.

PLoS One 2012 27;7(7):e40411. Epub 2012 Jul 27.

Research Centre, Novartis Vaccines and Diagnostics, Siena, Italy.

Streptococcus pyogenes is a major human pathogen worldwide, responsible for both local and systemic infections. These bacteria express the subtilisin-like protease SpyCEP which cleaves human IL-8 and related chemokines. We show that localization of SpyCEP is growth-phase and strain dependent. Significant shedding was observed only in a strain naturally overexpressing SpyCEP, and shedding was not dependent on SpyCEP autoproteolytic activity. Surface-bound SpyCEP in two different strains was capable of cleaving IL-8. To investigate SpyCEP action in vivo, we adapted the mouse air pouch model of infection for parallel quantification of bacterial growth, host immune cell recruitment and chemokine levels in situ. In response to infection, the predominant cells recruited were neutrophils, monocytes and eosinophils. Concomitantly, the chemokines KC, LIX, and MIP-2 in situ were drastically increased in mice infected with the SpyCEP knockout strain, and growth of this mutant strain was reduced compared to the wild type. SpyCEP has been described as a potential vaccine candidate against S. pyogenes, and we showed that surface-associated SpyCEP was recognized by specific antibodies. In vitro, such antibodies also counteracted the inhibitory effects of SpyCEP on chemokine mediated PMN recruitment. Thus, α-SpyCEP antibodies may benefit the host both directly by enabling opsonophagocytosis, and indirectly, by neutralizing an important virulence factor. The animal model we employed shows promise for broad application in the study of bacterial pathogenesis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0040411PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3407228PMC
April 2013

Staphylococcus aureus FhuD2 is involved in the early phase of staphylococcal dissemination and generates protective immunity in mice.

J Infect Dis 2012 Oct 24;206(7):1041-9. Epub 2012 Jul 24.

Novartis Vaccines, Research Center, Siena, Italy.

Iron availability plays an essential role in staphylococcal pathogenesis. We selected FhuD2, a lipoprotein involved in iron-hydroxamate uptake, as a novel vaccine candidate against Staphylococcus aureus. Unprecedented for staphylococcal lipoproteins, the protein was demonstrated to have a discrete, punctate localization on the bacterial surface. FhuD2 vaccination generated protective immunity against diverse clinical S. aureus isolates in murine infection models. Protection appeared to be associated with functional antibodies that were shown to mediate opsonophagocytosis, to be effective in passive transfer experiments, and to potentially block FhuD2-mediated siderophore uptake. Furthermore, the protein was found to be up-regulated in infected tissues and was required for staphylococcal dissemination and abscess formation. Herein we show that the staphylococcal iron-hydroxamate uptake system is important in invasive infection and functions as an efficacious vaccine target.
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http://dx.doi.org/10.1093/infdis/jis463DOI Listing
October 2012

The new multicomponent vaccine against meningococcal serogroup B, 4CMenB: immunological, functional and structural characterization of the antigens.

Vaccine 2012 May;30 Suppl 2:B87-97

Novartis Vaccines and Diagnostics, Via Fiorentina 1, 53100 Siena, Italy.

Neisseria meningitidis is a major cause of endemic cases and epidemics of meningitis and devastating septicemia. Although effective vaccines exist for several serogroups of pathogenic N. meningitidis, conventional vaccinology approaches have failed to provide a universal solution for serogroup B (MenB) which consequently remains an important burden of disease worldwide. The advent of whole-genome sequencing changed the approach to vaccine development, enabling the identification of potential vaccine candidates starting directly with the genomic information, with a process named reverse vaccinology. The application of reverse vaccinology to MenB allowed the identification of new protein antigens able to induce bactericidal antibodies. Three highly immunogenic antigens (fHbp, NadA and NHBA) were combined with outer membrane vesicles and formulated for human use in a multicomponent vaccine, named 4CMenB. This is the first MenB vaccine based on recombinant proteins able to elicit a robust bactericidal immune response in adults, adolescents and infants against a broad range of serogroup B isolates. This review describes the successful story of the development of the 4CMenB vaccine, with particular emphasis on the functional, immunological and structural characterization of the protein antigens included in the vaccine.
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http://dx.doi.org/10.1016/j.vaccine.2012.01.033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3360877PMC
May 2012

Future challenges in the elimination of bacterial meningitis.

Vaccine 2012 May;30 Suppl 2:B78-86

Novartis Vaccines & Diagnostics srl, Via Fiorentina 1, 53100 Siena, Italy.

Despite the widespread implementation of several effective vaccines over the past few decades, bacterial meningitis caused by Streptococcus pneumoniae, Haemophilus influenzae, Neisseria meningitidis and Group B Streptococcus (GBS) still results in unacceptably high levels of human mortality and morbidity. A residual disease burden due to bacterial meningitis is also apparent due to a number of persistent or emerging pathogens, including Mycobacterium tuberculosis, Escherichia coli, Staphylococcus aureus, Salmonella spp. and Streptococcus suis. Here, we review the current status of bacterial meningitis caused by these pathogens, highlighting how past and present vaccination programs have attempted to counter these pathogens. We discuss how improved pathogen surveillance, implementation of current vaccines, and development of novel vaccines may be expected to further reduce bacterial meningitis and related diseases in the future.
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http://dx.doi.org/10.1016/j.vaccine.2011.12.099DOI Listing
May 2012

Recombinant bacterial vaccines.

Curr Opin Immunol 2012 Jun 26;24(3):337-42. Epub 2012 Apr 26.

Novartis Vaccines and Diagnostics, Via Fiorentina 1, 53100, Siena, Italy.

Vaccines are currently available for many infectious diseases caused by several microbes and the prevention of disease and death by vaccination has profoundly improved public health globally. However, vaccines are not yet licensed for use against many other infectious diseases and new or improved vaccines are needed to replace suboptimal vaccines, and against newly emerging pathogens. Most of the vaccines currently licensed for human use include live attenuated and inactivated or killed microorganisms. Only a small subset is based on purified components and even fewer are recombinantly produced. Novel approaches in recombinant DNA technology, genomics and structural biology have revolutionized the way vaccine candidates are developed and will make a significant impact in the generation of safer and more effective vaccines.
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http://dx.doi.org/10.1016/j.coi.2012.03.013DOI Listing
June 2012

Structure of the C-terminal domain of Neisseria heparin binding antigen (NHBA), one of the main antigens of a novel vaccine against Neisseria meningitidis.

J Biol Chem 2011 Dec 29;286(48):41767-41775. Epub 2011 Sep 29.

MRC National Institute for Medical Research, The Ridgeway, London NW71AA, United Kingdom. Electronic address:

Neisseria heparin binding antigen (NHBA), also known as GNA2132 (genome-derived Neisseria antigen 2132), is a surface-exposed lipoprotein from Neisseria meningitidis that was originally identified by reverse vaccinology. It is one the three main antigens of a multicomponent vaccine against serogroup B meningitis (4CMenB), which has just completed phase III clinical trials in infants. In contrast to the other two main vaccine components, little is known about the origin of the immunogenicity of this antigen, and about its ability to induce a strong cross-bactericidal response in animals and humans. To characterize NHBA in terms of its structural/immunogenic properties, we have analyzed its sequence and identified a C-terminal region that is highly conserved in all strains. We demonstrate experimentally that this region is independently folded, and solved its three-dimensional structure by nuclear magnetic resonance. Notably, we need detergents to observe a single species in solution. The NHBA domain fold consists of an 8-strand β-barrel that closely resembles the C-terminal domains of N. meningitidis factor H-binding protein and transferrin-binding protein B. This common fold together with more subtle structural similarities suggest a common ancestor for these important antigens and a role of the β-barrel fold in inducing immunogenicity against N. meningitidis. Our data represent the first step toward understanding the relationship between structural, functional, and immunological properties of this important vaccine component.
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http://dx.doi.org/10.1074/jbc.M111.289314DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3308885PMC
December 2011

A naturally occurring single-residue mutation in the translocator domain of Neisseria meningitidis NhhA affects trimerization, surface localization, and adhesive capabilities.

Infect Immun 2011 Nov 15;79(11):4308-21. Epub 2011 Aug 15.

Novartis Vaccines and Diagnostics, Via Fiorentina, 1, 53100 Siena, Italy.

Neisseria meningitidis NhhA (Neisseria hia/hsf homologue A) is an oligomeric outer membrane protein belonging to the family of trimeric autotransporter adhesins. NhhA mediates the interaction of N. meningitidis with human epithelial cells and components of the extracellular matrix. The recombinant protein is able to induce bactericidal antibodies and hence has also been considered a potential vaccine candidate. In this study, we analyzed the production of NhhA in a large panel of N. meningitidis strains belonging to different serogroups and clonal complexes. We found that trimeric NhhA was produced at different levels by the various strains tested. In some strains belonging to the clonal complex ST41/44, the protein is detectable only as a monomer. Sequencing of the nhhA gene and generation of complementing strains in different genetic backgrounds have proved that a single mutation (Gly to Asp) in the translocator domain affected both trimerization and surface localization of NhhA. In vitro infection assays showed that this mutation impairs meningococcal NhhA-mediated adhesion, suggesting that strains carrying the mutation may rely on different strategies or molecules to mediate interaction with host cells. Finally, we demonstrated that N. meningitidis ST41/44 strains producing the mutated form did not induce killing mediated by NhhA-specific bactericidal antibodies. Our data help to elucidate the secretion mechanisms of trimeric autotransporters and to understand the contribution of NhhA in the evolutionary process of host-Neisseria interactions. Also, they might have important implications for the evaluation of NhhA as a vaccine candidate.
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http://dx.doi.org/10.1128/IAI.00198-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3257927PMC
November 2011

Transcriptome analysis of Neisseria meningitidis in human whole blood and mutagenesis studies identify virulence factors involved in blood survival.

PLoS Pathog 2011 May 5;7(5):e1002027. Epub 2011 May 5.

Novartis Vaccines and Diagnostics, Siena, Italy.

During infection Neisseria meningitidis (Nm) encounters multiple environments within the host, which makes rapid adaptation a crucial factor for meningococcal survival. Despite the importance of invasion into the bloodstream in the meningococcal disease process, little is known about how Nm adapts to permit survival and growth in blood. To address this, we performed a time-course transcriptome analysis using an ex vivo model of human whole blood infection. We observed that Nm alters the expression of ≈30% of ORFs of the genome and major dynamic changes were observed in the expression of transcriptional regulators, transport and binding proteins, energy metabolism, and surface-exposed virulence factors. In particular, we found that the gene encoding the regulator Fur, as well as all genes encoding iron uptake systems, were significantly up-regulated. Analysis of regulated genes encoding for surface-exposed proteins involved in Nm pathogenesis allowed us to better understand mechanisms used to circumvent host defenses. During blood infection, Nm activates genes encoding for the factor H binding proteins, fHbp and NspA, genes encoding for detoxifying enzymes such as SodC, Kat and AniA, as well as several less characterized surface-exposed proteins that might have a role in blood survival. Through mutagenesis studies of a subset of up-regulated genes we were able to identify new proteins important for survival in human blood and also to identify additional roles of previously known virulence factors in aiding survival in blood. Nm mutant strains lacking the genes encoding the hypothetical protein NMB1483 and the surface-exposed proteins NalP, Mip and NspA, the Fur regulator, the transferrin binding protein TbpB, and the L-lactate permease LctP were sensitive to killing by human blood. This increased knowledge of how Nm responds to adaptation in blood could also be helpful to develop diagnostic and therapeutic strategies to control the devastating disease cause by this microorganism.
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http://dx.doi.org/10.1371/journal.ppat.1002027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3088726PMC
May 2011

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

Proc Natl Acad Sci U S A 2011 Mar 28;108(11):4494-9. Epub 2011 Feb 28.

Research Center, Novartis Vaccines and Diagnostics, 53100 Siena, Italy.

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.
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http://dx.doi.org/10.1073/pnas.1019751108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3060241PMC
March 2011

A novel Hfq-dependent sRNA that is under FNR control and is synthesized in oxygen limitation in Neisseria meningitidis.

Mol Microbiol 2011 Apr 7;80(2):507-23. Epub 2011 Mar 7.

Novartis Vaccines, Microbial Molecular Biology, Via Fiorentina 1, 53100 Siena, Italy.

Small non-coding RNAs (sRNA) are emerging as key elements of post-transcriptional gene regulation in bacteria. The conserved Hfq protein is thought to function as an RNA chaperone and facilitate base-pairing between sRNAs and mRNA targets. In this study we identify a novel sRNA of Neisseria meningitidis through global gene expression studies of regulated transcripts in the Hfq mutant. The synthesis of this sRNA, named AniS, is anaerobically induced through activation of its promoter by the FNR global regulator. Whole-genome expression analyses led to the identification of putative mRNA targets, two of which are predicted to base pair with AniS. We show that Hfq binds the AniS transcript in vitro and is necessary for the downregulation of the identified target mRNAs in vivo. Contrary to many Hfq-dependent sRNA of the Enterobacteriaceae, Hfq promotes decay of AniS in N. meningitidis. Our analysis shows that the AniS regulator is part of the FNR regulon and may be responsible for the downregulation of FNR-repressed genes. Furthermore the presence of similar conserved regulatory sequences in all Neisseria spp. to date suggests that an analogous FNR-regulated sRNA, with a variable 5' sequence, may be ubiquitous to all commensals and pathogens of the Genus.
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http://dx.doi.org/10.1111/j.1365-2958.2011.07592.xDOI Listing
April 2011

Influence of sequence variability on bactericidal activity sera induced by Factor H binding protein variant 1.1.

Vaccine 2011 Jan 3;29(5):1072-81. Epub 2010 Dec 3.

Novartis Vaccines and Diagnostics, Via Fiorentina 1, 53100 Siena, Italy.

Factor H binding protein (fHbp), one of the main antigens of new vaccines against serogroup B meningococcus, varies in amino acid sequence and level of expression in different clinical isolates. To evaluate the contribution of amino acid sequence variability to vaccine coverage, we constructed a strain that is susceptible to bactericidal killing only by anti-fHbp antibodies and engineered it to express equal levels of 10 different fHbp sub-variants from a constitutive promoter. Testing of these isogenic strains showed that sera from mice or adult volunteers vaccinated with fHbp variant 1.1 were bactericidal against all sub-variants 1 sequences, however the titer against the most distant sequences were several times lower. Sera from vaccinated infants were more susceptible to amino acid variations and they had lower or no bactericidal activity against the distant sub-variants 1 sequences in comparison with sera from adults given the same vaccines. The low coverage provided by fHbp could be overcome using a multicomponent vaccine. We conclude that fHbp is a very important antigen that induces bactericidal antibodies in animals, adults and infants. However, given its high variability of sequence and expression level, it is unlikely that fHbp alone can provide good protection in infants against the distant amino acid sequence variants and therefore multicomponent vaccines inducing protective immunity also against other antigens are more likely to induce a broad protective immunity in all age groups.
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http://dx.doi.org/10.1016/j.vaccine.2010.11.064DOI Listing
January 2011

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

Proc Natl Acad Sci U S A 2010 Nov 20;107(45):19490-5. Epub 2010 Oct 20.

Novartis Vaccines and Diagnostics, 53100 Siena, Italy.

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.
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http://dx.doi.org/10.1073/pnas.1013758107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2984153PMC
November 2010

Measuring antigen-specific bactericidal responses to a multicomponent vaccine against serogroup B meningococcus.

Vaccine 2010 Jul 21;28(31):5023-30. Epub 2010 May 21.

Novartis Vaccines and Diagnostics, Via Fiorentina 1, Siena 53100, Italy.

Serum bactericidal activity using human complement is the basis for established correlates of protection against invasive meningococcal disease. During the development of multicomponent protein-based vaccines against meningococcus B, it is necessary to measure antigen-specific bactericidal responses. This is not straightforward because each strain may be killed by antibodies to multiple antigens. We characterized a large panel of strains and, using a competitive inhibition SBA, we identified four strains that are each specifically killed by bactericidal antibodies to one of the major vaccine components. These strains provide a straightforward approach to demonstrate protective responses to each component of the vaccine and demonstrate that each of the antigens in the vaccine is sufficient to provide a potentially protective level of bactericidal activity.
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http://dx.doi.org/10.1016/j.vaccine.2010.05.014DOI Listing
July 2010

Molecular mechanisms of complement evasion: learning from staphylococci and meningococci.

Nat Rev Microbiol 2010 06;8(6):393-9

Novartis Vaccines and Diagnostics, Via Fiorentina 1, 53100 Siena, Italy.

The complement system is a crucial component of the innate immune response in humans. Recent studies in Staphylococcus aureus and Neisseria meningitidis have revealed how these bacteria escape complement-mediated killing. In addition, new structural data have provided detailed insights into the molecular mechanisms of host defence mediated by the complement system and how bacterial proteins interfere with this process. This information is fundamental to our understanding of bacterial pathogenesis and may facilitate the design of better vaccines.
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http://dx.doi.org/10.1038/nrmicro2366DOI Listing
June 2010

Neisseria meningitidis GNA2132, a heparin-binding protein that induces protective immunity in humans.

Proc Natl Acad Sci U S A 2010 Feb 3;107(8):3770-5. Epub 2010 Feb 3.

Novartis Vaccines and Diagnostics, 53100 Siena, Italy.

GNA2132 is a Neisseria meningitidis antigen of unknown function, discovered by reverse vaccinology, which has been shown to induce bactericidal antibodies in animal models. Here we show that this antigen induces protective immunity in humans and it is recognized by sera of patients after meningococcal disease. The protein binds heparin in vitro through an Arg-rich region and this property correlates with increased survival of the unencapsulated bacterium in human serum. Furthermore, two proteases, the meningococcal NalP and human lactoferrin, cleave the protein upstream and downstream from the Arg-rich region, respectively. We conclude that GNA2132 is an important protective antigen of N. meningitidis and we propose to rename it, Neisserial Heparin Binding Antigen (NHBA).
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http://dx.doi.org/10.1073/pnas.0915162107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2840514PMC
February 2010

HadA is an atypical new multifunctional trimeric coiled-coil adhesin of Haemophilus influenzae biogroup aegyptius, which promotes entry into host cells.

Cell Microbiol 2009 Jul 12;11(7):1044-63. Epub 2009 Mar 12.

Research Center, Novartis Vaccines and Diagnostics, Siena, Italy.

The Oca (Oligomeric coiled-coil adhesin) family is a subgroup of the bacterial trimeric autotransporter adhesins, which includes structurally related proteins, such as YadA of Yersinia enterocolitica and NadA of Neisseria meningitidis. In this study, we searched in silico for novel members of this family in bacterial genomes and identified HadA (Haemophilus adhesin A), a trimeric autotransporter expressed only by Haemophilus influenzae biogroup aegyptius causing Brazilian purpuric fever (BPF), a fulminant septicemic disease of children. By comparative genomics and sequence analysis we predicted that the hadA gene is harboured on a mobile genetic element unique to BPF isolates. Biological analysis of HadA in the native background was limited because this organism is not amenable to genetic manipulation. Alternatively, we demonstrated that expression of HadA confers to a non-invasive Escherichia coli strain the ability to adhere to human cells and to extracellular matrix proteins and to induce in vitro bacterial aggregation and microcolony formation. Intriguingly, HadA is predicted to lack the typical N-terminal head domain of Oca proteins generally associated with cellular receptor binding. We propose here a structural model of the HadA coiled-coil stalk and show that the N-terminal region is still responsible of the binding activity and a KGD motif plays a role. Interestingly, HadA promotes bacterial entry into mammalian cells. Our results show a cytoskeleton re-arrangement and an involvement of clathrin in the HadA-mediated internalization. These data give new insights on the structure-function relationship of oligomeric coiled-coil adhesins and suggest a potential role of this protein in the pathogenesis of BPF.
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http://dx.doi.org/10.1111/j.1462-5822.2009.01306.xDOI Listing
July 2009

Genome-based approaches to develop vaccines against bacterial pathogens.

Vaccine 2009 May 5;27(25-26):3245-50. Epub 2009 Feb 5.

Novartis Vaccines and Diagnostics, Via Fiorentina 1, 53100 Siena, Italy.

Bacterial infectious diseases remain the single most important threat to health worldwide. Although conventional vaccinology approaches were successful in conferring protection against several diseases, they failed to provide efficacious solutions against many others. The advent of whole-genome sequencing changed the way to think about vaccine development, enabling the targeting of possible vaccine candidates starting from the genomic information of a single bacterial isolate, with a process named reverse vaccinology. As the genomic era progressed, reverse vaccinology has evolved with a pan-genome approach and multi-strain genome analysis became fundamental for the design of universal vaccines. This review describes the applications of genome-based approaches in the development of new vaccines against bacterial pathogens.
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http://dx.doi.org/10.1016/j.vaccine.2009.01.072DOI Listing
May 2009