Publications by authors named "Silvana Savino"

37 Publications

Lectin activity of Pseudomonas aeruginosa vaccine candidates PSE17-1, PSE41-5 and PSE54.

Biochem Biophys Res Commun 2019 05 3;513(1):287-290. Epub 2019 Apr 3.

Institute for Glycomics, Griffith University, Gold Coast, QLD, 4222, Australia. Electronic address:

Pseudomonas aeruginosa is an opportunistic pathogen that causes nosocomial infections most commonly in immunocompromised, cystic fibrosis (CF) and burns patients. The pilin and Pseudomonas lectins 1 (PA-IL) and 2 (PA-IIL) are known glycan-binding proteins of P. aeruginosa that are involved in adherence to host cells, particularly CF host airways. Recently, new P. aeruginosa surface proteins were identified by reverse vaccinology and tested in vivo as potential vaccine antigens. Three of these, namely PSE17-1, PSE41-5 and PSE54, were screened for glycan binding using glycan arrays displaying glycan structures representative of those found on human cells. Surface plasmon resonance was used to confirm the lectin activity of these proteins, and determined affinities with several host glycans to be in the nanomolar range. PSE17-1 binds hyaluronic acid and sialyl Lewis A and X. PSE41-5 binds terminal β-linked galactose structures, Lewis and ABO blood group antigens. PSE54 binds to ABO blood group antigens and some terminal β-linked galactose. All three proteins are novel lectins of P. aeruginosa with potential roles in infection of host cells.
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http://dx.doi.org/10.1016/j.bbrc.2019.03.092DOI Listing
May 2019

Lectin Activity of the TcdA and TcdB Toxins of Clostridium difficile.

Infect Immun 2019 03 21;87(3). Epub 2019 Feb 21.

Institute for Glycomics, Griffith University, Gold Coast, Australia

is a major cause of hospital-acquired antibiotic-associated diarrhea. produces two cytotoxins, TcdA and TcdB; both toxins are multidomain proteins that lead to cytotoxicity through the modification and inactivation of small GTPases of the Rho/Rac family. Previous studies have indicated that host glycans are targets for TcdA and TcdB, with interactions thought to be with both α- and β-linked galactose. In the current study, screening of glycan arrays with different domains of TcdA and TcdB revealed that the binding regions of both toxins interact with a wider range of host glycoconjugates than just terminal α- and β-linked galactose, including blood groups, Lewis antigens, -acetylglucosamine, mannose, and glycosaminoglycans. The interactions of TcdA and TcdB with ABO blood group and Lewis antigens were assessed by surface plasmon resonance (SPR). The blood group A antigen was the highest-affinity ligand for both toxins. Free glycans alone or in combination were unable to abolish Vero cell cytotoxicity by TcdB. SPR competition assays indicate that there is more than one glycan binding site on TcdB. Host glycoconjugates are common targets of bacterial toxins, but typically this binding is to a specific structure or related structures. The binding of TcdA and TcdB is to a wide range of host glycans providing a wide range of target cells and tissues .
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http://dx.doi.org/10.1128/IAI.00676-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6386544PMC
March 2019

Lectin activity of the pneumococcal pilin proteins.

Sci Rep 2017 12 19;7(1):17784. Epub 2017 Dec 19.

Institute for Glycomics, Griffith University, Gold Coast, QLD 4222, Australia.

Streptococcus pneumoniae is a leading cause of morbidity and mortality globally. The Pilus-1 proteins, RrgA, RrgB and RrgC of S. pneumoniae have been previously assessed for their role in infection, invasive disease and as possible vaccine candidates. In this study we have investigated the glycan binding repertoire of all three Pilus-1 proteins, identifying that the tip adhesin RrgA has the broadest glycan recognition of the three proteins, binding to maltose/cellobiose, α/β linked galactose and blood group A and H antigens. RrgB only bound mannose, while RrgC bound a subset of glycans also recognized by RrgA. Adherence of S. pneumoniae TIGR4 to epithelial cells was tested using four of the oligosaccharides identified through the glycan array analysis as competitive inhibitors. The blood group H trisaccharide provided the best blocking of S. pneumoniae TIGR4 adherence. Adherence is the first step in disease, and host glycoconjugates are a common target for many adhesins. This study has identified Pilus-1 proteins as new lectins involved in the targeting of host glycosylation by S. pneumoniae.
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http://dx.doi.org/10.1038/s41598-017-17850-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5736695PMC
December 2017

Exploring host-pathogen interactions through genome wide protein microarray analysis.

Sci Rep 2016 06 15;6:27996. Epub 2016 Jun 15.

GSK Vaccines, Via Fiorentina 1, 53100 Siena, Italy.

During bacterial pathogenesis extensive contacts between the human and the bacterial extracellular proteomes take place. The identification of novel host-pathogen interactions by standard methods using a case-by-case approach is laborious and time consuming. To overcome this limitation, we took advantage of large libraries of human and bacterial recombinant proteins. We applied a large-scale protein microarray-based screening on two important human pathogens using two different approaches: (I) 75 human extracellular proteins were tested on 159 spotted Staphylococcus aureus recombinant proteins and (II) Neisseria meningitidis adhesin (NadA), an important vaccine component against serogroup B meningococcus, was screened against ≈2300 spotted human recombinant proteins. The approach presented here allowed the identification of the interaction between the S. aureus immune evasion protein FLIPr (formyl-peptide receptor like-1 inhibitory protein) and the human complement component C1q, key players of the offense-defense fighting; and of the interaction between meningococcal NadA and human LOX-1 (low-density oxidized lipoprotein receptor), an endothelial receptor. The novel interactions between bacterial and human extracellular proteins here presented might provide a better understanding of the molecular events underlying S. aureus and N. meningitidis pathogenesis.
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http://dx.doi.org/10.1038/srep27996DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4908583PMC
June 2016

Expression of factor H binding protein in meningococcal strains can vary at least 15-fold and is genetically determined.

Proc Natl Acad Sci U S A 2016 Mar 17;113(10):2714-9. Epub 2016 Feb 17.

GSK Vaccines, 53100 Siena, Italy

Factor H binding protein (fHbp) is a lipoprotein of Neisseria meningitidis important for the survival of the bacterium in human blood and a component of two recently licensed vaccines against serogroup B meningococcus (MenB). Based on 866 different amino acid sequences this protein is divided into three variants or two families. Quantification of the protein is done by immunoassays such as ELISA or FACS that are susceptible to the sequence variation and expression level of the protein. Here, selected reaction monitoring mass spectrometry was used for the absolute quantification of fHbp in a large panel of strains representative of the population diversity of MenB. The analysis revealed that the level of fHbp expression can vary at least 15-fold and that variant 1 strains express significantly more protein than variant 2 or variant 3 strains. The susceptibility to complement-mediated killing correlated with the amount of protein expressed by the different meningococcal strains and this could be predicted from the nucleotide sequence of the promoter region. Finally, the absolute quantification allowed the calculation of the number of fHbp molecules per cell and to propose a mechanistic model of the engagement of C1q, the recognition component of the complement cascade.
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http://dx.doi.org/10.1073/pnas.1521142113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4791009PMC
March 2016

Recognition of Neisseria meningitidis by the long pentraxin PTX3 and its role as an endogenous adjuvant.

PLoS One 2015 18;10(3):e0120807. Epub 2015 Mar 18.

Department of Inflammation and Immunology, Humanitas Clinical and Research Center, Rozzano (Milan), Italy; Humanitas University, Rozzano (Milan), Italy.

Long pentraxin 3 (PTX3) is a non-redundant component of the humoral arm of innate immunity. The present study was designed to investigate the interaction of PTX3 with Neisseria meningitidis. PTX3 bound acapsular meningococcus, Neisseria-derived outer membrane vesicles (OMV) and 3 selected meningococcal antigens (GNA0667, GNA1030 and GNA2091). PTX3-recognized microbial moieties are conserved structures which fulfil essential microbial functions. Ptx3-deficient mice had a lower antibody response in vaccination protocols with OMV and co-administration of PTX3 increased the antibody response, particularly in Ptx3-deficient mice. Administration of PTX3 reduced the bacterial load in infant rats challenged with Neisseria meningitidis. These results suggest that PTX3 recognizes a set of conserved structures from Neisseria meningitidis and acts as an amplifier/endogenous adjuvant of responses to this bacterium.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0120807PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4364741PMC
February 2016

Vaccine composition formulated with a novel TLR7-dependent adjuvant induces high and broad protection against Staphylococcus aureus.

Proc Natl Acad Sci U S A 2015 Mar 9;112(12):3680-5. Epub 2015 Mar 9.

Novartis Vaccines Research Center, 53100 Siena, Italy;

Both active and passive immunization strategies against Staphylococcus aureus have thus far failed to show efficacy in humans. With the attempt to develop an effective S. aureus vaccine, we selected five conserved antigens known to have different roles in S. aureus pathogenesis. They include the secreted factors α-hemolysin (Hla), ess extracellular A (EsxA), and ess extracellular B (EsxB) and the two surface proteins ferric hydroxamate uptake D2 and conserved staphylococcal antigen 1A. The combined vaccine antigens formulated with aluminum hydroxide induced antibodies with opsonophagocytic and functional activities and provided consistent protection in four mouse models when challenged with a panel of epidemiologically relevant S. aureus strains. The importance of antibodies in protection was demonstrated by passive transfer experiments. Furthermore, when formulated with a toll-like receptor 7-dependent (TLR7) agonist recently designed and developed in our laboratories (SMIP.7-10) adsorbed to alum, the five antigens provided close to 100% protection against four different staphylococcal strains. The new formulation induced not only high antibody titers but also a Th1 skewed immune response as judged by antibody isotype and cytokine profiles. In addition, low frequencies of IL-17-secreting T cells were also observed. Altogether, our data demonstrate that the rational selection of mixtures of conserved antigens combined with Th1/Th17 adjuvants can lead to promising vaccine formulations against S. aureus.
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http://dx.doi.org/10.1073/pnas.1424924112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4378396PMC
March 2015

Structure of the meningococcal vaccine antigen NadA and epitope mapping of a bactericidal antibody.

Proc Natl Acad Sci U S A 2014 Dec 17;111(48):17128-33. Epub 2014 Nov 17.

Novartis Vaccines, 53100 Siena, Italy; and.

Serogroup B Neisseria meningitidis (MenB) is a major cause of severe sepsis and invasive meningococcal disease, which is associated with 5-15% mortality and devastating long-term sequelae. Neisserial adhesin A (NadA), a trimeric autotransporter adhesin (TAA) that acts in adhesion to and invasion of host epithelial cells, is one of the three antigens discovered by genome mining that are part of the MenB vaccine that recently was approved by the European Medicines Agency. Here we present the crystal structure of NadA variant 5 at 2 Å resolution and transmission electron microscopy data for NadA variant 3 that is present in the vaccine. The two variants show similar overall topology with a novel TAA fold predominantly composed of trimeric coiled-coils with three protruding wing-like structures that create an unusual N-terminal head domain. Detailed mapping of the binding site of a bactericidal antibody by hydrogen/deuterium exchange MS shows that a protective conformational epitope is located in the head of NadA. These results provide information that is important for elucidating the biological function and vaccine efficacy of NadA.
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http://dx.doi.org/10.1073/pnas.1419686111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4260552PMC
December 2014

Role of ARF6, Rab11 and external Hsp90 in the trafficking and recycling of recombinant-soluble Neisseria meningitidis adhesin A (rNadA) in human epithelial cells.

PLoS One 2014 27;9(10):e110047. Epub 2014 Oct 27.

Novartis Vaccines, Siena, Italy; Department of Biology, University of Naples "Federico II", Naples, Italy.

Neisseria meningitidis adhesin A (NadA) is a meningococcus surface protein thought to assist in the adhesion of the bacterium to host cells. We have previously shown that NadA also promotes bacterial internalization in a heterologous expression system. Here we have used the soluble recombinant NadA (rNadA) lacking the membrane anchor region to characterize its internalization route in Chang epithelial cells. Added to the culture medium, rNadA internalizes through a PI3K-dependent endocytosis process not mediated by the canonical clathrin or caveolin scaffolds, but instead follows an ARF6-regulated recycling pathway previously described for MHC-I. The intracellular pool of rNadA reaches a steady state level within one hour of incubation and colocalizes in endocytic vesicles with MHC-I and with the extracellularly labeled chaperone Hsp90. Treatment with membrane permeated and impermeable Hsp90 inhibitors 17-AAG and FITC-GA respectively, lead to intracellular accumulation of rNadA, strongly suggesting that the extracellular secreted pool of the chaperone is involved in rNadA intracellular trafficking. A significant number of intracellular vesicles containing rNadA recruit Rab11, a small GTPase associated to recycling endosomes, but do not contain transferrin receptor (TfR). Interestingly, cell treatment with Hsp90 inhibitors, including the membrane-impermeable FITC-GA, abolished Rab11-rNadA colocalization but do not interfere with Rab11-TfR colocalization. Collectively, these results are consistent with a model whereby rNadA internalizes into human epithelial cells hijacking the recycling endosome pathway and recycle back to the surface of the cell via an ARF6-dependent, Rab11 associated and Hsp90-regulated mechanism. The present study addresses for the first time a meningoccoccal adhesin mechanism of endocytosis and suggests a possible entry pathway engaged by N. meningitidis in primary infection of human epithelial cells.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0110047PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4210143PMC
June 2015

Structure and protective efficacy of the Staphylococcus aureus autocleaving protease EpiP.

FASEB J 2014 Apr 13;28(4):1780-93. Epub 2014 Jan 13.

2G.G., Novartis Vaccines, via Fiorentina 1, 53100, Siena, Italy.

Despite the global medical needs associated with Staphylococcus aureus infections, no licensed vaccines are currently available. We identified and characterized a protein annotated as an epidermin leader peptide processing serine protease (EpiP), as a novel S. aureus vaccine candidate. In addition, we determined the structure of the recombinant protein (rEpiP) by X-ray crystallography. The crystal structure revealed that rEpiP was cleaved somewhere between residues 95 and 100, and we found that the cleavage occurs through an autocatalytic intramolecular mechanism. The protein expressed by S. aureus cells also appeared to undergo a similar processing event. To determine whether the protein acts as a serine protease, we mutated the hypothesized catalytic serine 393 residue to alanine, generating rEpiP-S393A. The crystal structure of this mutant protein showed that the polypeptide chain was not cleaved and was not interacting stably with the active site. Indeed, rEpiP-S393A was shown to be impaired in its protease activity. Mice vaccinated with rEpiP were protected from S. aureus infection (34% survival, P=0.0054). Moreover, the protective efficacy generated by rEpiP and rEpiP-S393A was comparable, implying that the noncleaving mutant could be used for vaccination purposes.
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http://dx.doi.org/10.1096/fj.13-241737DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3963019PMC
April 2014

Two cross-reactive monoclonal antibodies recognize overlapping epitopes on Neisseria meningitidis factor H binding protein but have different functional properties.

FASEB J 2014 Apr 26;28(4):1644-53. Epub 2013 Dec 26.

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

Factor H binding protein (fHbp) is one of the main antigens of the 4-component meningococcus B (4CMenB) multicomponent vaccine against disease caused by serogroup B Neisseria meningitidis (MenB). fHbp binds the complement down-regulating protein human factor H (hfH), thus resulting in immune evasion. fHbp exists in 3 variant groups with limited cross-protective responses. Previous studies have described the generation of monoclonal antibodies (mAbs) targeting variant-specific regions of fHbp. Here we report for the first time the functional characterization of two mAbs that recognize a wide panel of fHbp variants and subvariants on the MenB surface and that are able to inhibit fHbp binding to hfH. The antigenic regions targeted by the two mAbs were accurately mapped by hydrogen-deuterium exchange mass spectrometry (HDX-MS), revealing partially overlapping epitopes on the N terminus of fHbp. Furthermore, while none of the mAbs had bactericidal activity on its own, a synergistic effect was observed for each of them when tested by the human complement serum bactericidal activity (hSBA) assay in combination with a second nonbactericidal mAb. The bases underlying fHbp variant cross-reactivity, as well as inhibition of hfH binding and cooperativity effect observed for the two mAbs, are discussed in light of the mapped epitopes.
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http://dx.doi.org/10.1096/fj.13-239012DOI Listing
April 2014

Protective efficacy induced by recombinant Clostridium difficile toxin fragments.

Infect Immun 2013 Aug 28;81(8):2851-60. Epub 2013 May 28.

Novartis Vaccines and Diagnostics SRL, Siena, Italy.

Clostridium difficile is a spore-forming bacterium that can reside in animals and humans. C. difficile infection causes a variety of clinical symptoms, ranging from diarrhea to fulminant colitis. Disease is mediated by TcdA and TcdB, two large enterotoxins released by C. difficile during colonization of the gut. In this study, we evaluated the ability of recombinant toxin fragments to induce neutralizing antibodies in mice. The protective efficacies of the most promising candidates were then evaluated in a hamster model of disease. While limited protection was observed with some combinations, coadministration of a cell binding domain fragment of TcdA (TcdA-B1) and the glucosyltransferase moiety of TcdB (TcdB-GT) induced systemic IgGs which neutralized both toxins and protected vaccinated animals from death following challenge with two strains of C. difficile. Further characterization revealed that despite high concentrations of toxin in the gut lumens of vaccinated animals during the acute phase of the disease, pathological damage was minimized. Assessment of gut contents revealed the presence of TcdA and TcdB antibodies, suggesting that systemic vaccination with this pair of recombinant polypeptides can limit the disease caused by toxin production during C. difficile infection.
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http://dx.doi.org/10.1128/IAI.01341-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3719595PMC
August 2013

Defining a protective epitope on factor H binding protein, a key meningococcal virulence factor and vaccine antigen.

Proc Natl Acad Sci U S A 2013 Feb 8;110(9):3304-9. Epub 2013 Feb 8.

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

Mapping of epitopes recognized by functional monoclonal antibodies (mAbs) is essential for understanding the nature of immune responses and designing improved vaccines, therapeutics, and diagnostics. In recent years, identification of B-cell epitopes targeted by neutralizing antibodies has facilitated the design of peptide-based vaccines against highly variable pathogens like HIV, respiratory syncytial virus, and Helicobacter pylori; however, none of these products has yet progressed into clinical stages. Linear epitopes identified by conventional mapping techniques only partially reflect the immunogenic properties of the epitope in its natural conformation, thus limiting the success of this approach. To investigate antigen-antibody interactions and assess the potential of the most common epitope mapping techniques, we generated a series of mAbs against factor H binding protein (fHbp), a key virulence factor and vaccine antigen of Neisseria meningitidis. The interaction of fHbp with the bactericidal mAb 12C1 was studied by various epitope mapping methods. Although a 12-residue epitope in the C terminus of fHbp was identified by both Peptide Scanning and Phage Display Library screening, other approaches, such as hydrogen/deuterium exchange mass spectrometry (MS) and X-ray crystallography, showed that mAb 12C1 occupies an area of ∼1,000 Å(2) on fHbp, including >20 fHbp residues distributed on both N- and C-terminal domains. Collectively, these data show that linear epitope mapping techniques provide useful but incomplete descriptions of B-cell epitopes, indicating that increased efforts to fully characterize antigen-antibody interfaces are required to understand and design effective immunogens.
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http://dx.doi.org/10.1073/pnas.1222845110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3587270PMC
February 2013

The factor H binding protein of Neisseria meningitidis interacts with xenosiderophores in vitro.

Biochemistry 2012 Nov 12;51(46):9384-93. Epub 2012 Nov 12.

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

The factor H binding protein (fHbp) is a key virulence factor of Neisseria meningitidis that confers to the bacterium the ability to resist killing by human serum. The determination of its three-dimensional structure revealed that the carboxyl terminus of the protein folds into an eight-stranded β barrel. The structural similarity of this part of the protein to lipocalins provided the rationale for exploring the ability of fHbp to bind siderophores. We found that fHbp was able to bind in vitro siderophores belonging to the cathecolate family and mapped the interaction site by nuclear magnetic resonance. Our results indicated that the enterobactin binding site was distinct from the site involved in binding to human factor H and stimulates new hypotheses about possible multiple activities of fHbp.
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http://dx.doi.org/10.1021/bi301161wDOI Listing
November 2012

Structural and functional characterization of the Staphylococcus aureus virulence factor and vaccine candidate FhuD2.

Biochem J 2013 Feb;449(3):683-93

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

Staphylococcus aureus is a human pathogen causing globally significant morbidity and mortality. The development of antibiotic resistance in S. aureus highlights the need for a preventive vaccine. In the present paper we explore the structure and function of FhuD2 (ferric-hydroxamate uptake D2), a staphylococcal surface lipoprotein mediating iron uptake during invasive infection, recently described as a promising vaccine candidate. Differential scanning fluorimetry and calorimetry studies revealed that FhuD2 is stabilized by hydroxamate siderophores. The FhuD2-ferrichrome interaction was of nanomolar affinity in surface plasmon resonance experiments and fully iron(III)-dependent. We determined the X-ray crystallographic structure of ligand-bound FhuD2 at 1.9 Å (1 Å=0.1 nm) resolution, revealing the bilobate fold of class III SBPs (solute-binding proteins). The ligand, ferrichrome, occupies a cleft between the FhuD2 N- and C-terminal lobes. Many FhuD2-siderophore interactions enable the specific recognition of ferrichrome. Biochemical data suggest that FhuD2 does not undergo significant conformational changes upon siderophore binding, supporting the hypothesis that the ligand-bound complex is essential for receptor engagement and uptake. Finally, immunizations with FhuD2 alone or FhuD2 formulated with hydroxamate siderophores were equally protective in a murine staphylococcal infection model, confirming the suitability and efficacy of apo-FhuD2 as a protective antigen, and suggesting that other class III SBPs might also be exploited as vaccine candidates.
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http://dx.doi.org/10.1042/BJ20121426DOI Listing
February 2013

Development of an influenza virus protein array using Sortagging technology.

Bioconjug Chem 2012 Jun 25;23(6):1119-26. Epub 2012 May 25.

Whitehead Institute for Biomedical Research , 9 Cambridge Center, Cambridge, Massachusetts 02142, United States.

Protein array technology is an emerging tool that enables high-throughput screening of protein-protein or protein-lipid interactions and identification of immunodominant antigens during the course of a bacterial or viral infection. In this work, we developed an Influenza virus protein array using the sortase-mediated transpeptidation reaction known as "Sortagging". LPETG-tagged Influenza virus proteins from bacterial and eukaryotic cellular extracts were immobilized at their carboxyl-termini onto a preactivated amine-glass slide coated with a Gly3 linker. Immobilized proteins were revealed by specific antibodies, and the newly generated Sortag-protein chip can be used as a device for antigen and/or antibody screening. The specificity of the Sortase A (SrtA) reaction avoids purification steps in array building and allows immobilization of proteins in an oriented fashion. Previously, this versatile technology has been successfully employed for protein labeling and protein conjugation. Here, the tool is implemented to covalently link proteins of a viral genome onto a solid support. The system could readily be scaled up to proteins of larger genomes in order to develop protein arrays for high-throughput screening.
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http://dx.doi.org/10.1021/bc200577uDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3884033PMC
June 2012

RrgB321, a fusion protein of the three variants of the pneumococcal pilus backbone RrgB, is protective in vivo and elicits opsonic antibodies.

Infect Immun 2012 Jan 14;80(1):451-60. Epub 2011 Nov 14.

Research Center, Novartis Vaccines and Diagnostics s.r.l., Siena, Italy.

Streptococcus pneumoniae pilus 1 is present in 30 to 50% of invasive disease-causing strains and is composed of three subunits: the adhesin RrgA, the major backbone subunit RrgB, and the minor ancillary protein RrgC. RrgB exists in three distinct genetic variants and, when used to immunize mice, induces an immune response specific for each variant. To generate an antigen able to protect against the infection caused by all pilus-positive S. pneumoniae strains, we engineered a fusion protein containing the three RrgB variants (RrgB321). RrgB321 elicited antibodies against proteins from organisms in the three clades and protected mice against challenge with piliated pneumococcal strains. RrgB321 antisera mediated complement-dependent opsonophagocytosis of piliated strains at levels comparable to those achieved with the PCV7 glycoconjugate vaccine. These results suggest that a vaccine composed of RrgB321 has the potential to cover 30% or more of all pneumococcal strains and support the inclusion of this fusion protein in a multicomponent vaccine against S. pneumoniae.
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http://dx.doi.org/10.1128/IAI.05780-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3255673PMC
January 2012

Antigen identification starting from the genome: a "Reverse Vaccinology" approach applied to MenB.

Methods Mol Biol 2012 ;799:361-403

Novartis Vaccines and Diagnostics, Siena, Italy.

Most of the vaccines available today, albeit very effective, have been developed using traditional "old-style" methodologies. Technologies developed in recent years have opened up new perspectives in the field of vaccinology and novel strategies are now being used to design improved or new vaccines against infections for which preventive measures do not exist. The Reverse Vaccinology (RV) approach is one of the most powerful examples of biotechnology applied to the field of vaccinology for identifying new protein-based vaccines. RV combines the availability of genomic data, the analyzing capabilities of new bioinformatic tools, and the application of high throughput expression and purification systems combined with serological screening assays for a coordinated screening process of the entire genomic repertoire of bacterial, viral, or parasitic pathogens. The application of RV to Neisseria meningitidis serogroup B represents the first success of this novel approach. In this chapter, we describe how this revolutionary approach can be easily applied to any pathogen.
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http://dx.doi.org/10.1007/978-1-61779-346-2_21DOI Listing
February 2012

Rational design of a meningococcal antigen inducing broad protective immunity.

Sci Transl Med 2011 Jul;3(91):91ra62

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

The sequence variability of protective antigens is a major challenge to the development of vaccines. For Neisseria meningitidis, the bacterial pathogen that causes meningitis, the amino acid sequence of the protective antigen factor H binding protein (fHBP) has more than 300 variations. These sequence differences can be classified into three distinct groups of antigenic variants that do not induce cross-protective immunity. Our goal was to generate a single antigen that would induce immunity against all known sequence variants of N. meningitidis. To achieve this, we rationally designed, expressed, and purified 54 different mutants of fHBP and tested them in mice for the induction of protective immunity. We identified and determined the crystal structure of a lead chimeric antigen that was able to induce high levels of cross-protective antibodies in mice against all variant strains tested. The new fHBP antigen had a conserved backbone that carried an engineered surface containing specificities for all three variant groups. We demonstrate that the structure-based design of multiple immunodominant antigenic surfaces on a single protein scaffold is possible and represents an effective way to create broadly protective vaccines.
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http://dx.doi.org/10.1126/scitranslmed.3002234DOI Listing
July 2011

Characterization of diverse subvariants of the meningococcal factor H (fH) binding protein for their ability to bind fH, to mediate serum resistance, and to induce bactericidal antibodies.

Infect Immun 2011 Feb 13;79(2):970-81. Epub 2010 Dec 13.

Molecular Genetics Unit, Novartis Vaccines, Via Fiorentina, 1, 53100 Siena, Italy.

Neisseria meningitidis is a commensal of the human nasopharynx but is also a major cause of septicemia and meningitis. The meningococcal factor H binding protein (fHbp) binds human factor H (fH), enabling downregulation of complement activation on the bacterial surface. fHbp is a component of two serogroup B meningococcal vaccines currently in clinical development. Here we characterize 12 fHbp subvariants for their level of surface exposure and ability to bind fH, to mediate serum resistance, and to induce bactericidal antibodies. Flow cytometry and Western analysis revealed that all strains examined expressed fHbp on their surface to different extents and bound fH in an fHbp-dependent manner. However, differences in fH binding did not always correlate with the level of fHbp expression, indicating that this is not the only factor affecting the amount of fH bound. To overcome the issue of strain variability in fHbp expression, the MC58ΔfHbp strain was genetically engineered to express different subvariants from a constitutive heterologous promoter. These recombinant strains were characterized for fH binding, and the data confirmed that each subvariant binds different levels of fH. Surface plasmon resonance revealed differences in the stability of the fHbp-fH complexes that ranged over 2 orders of magnitude, indicating that differences in residues between and within variant groups can influence fH binding. Interestingly, the level of survival in human sera of recombinant MC58 strains expressing diverse subvariants did not correlate with the level of fH binding, suggesting that the interaction of fHbp with fH is not the only function of fHbp that influences serum resistance. Furthermore, cross-reactive bactericidal activity was seen within each variant group, although the degree of activity varied, suggesting that amino acid differences within each variant group influence the bactericidal antibody response.
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http://dx.doi.org/10.1128/IAI.00891-10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3028832PMC
February 2011

Identification of protective and broadly conserved vaccine antigens from the genome of extraintestinal pathogenic Escherichia coli.

Proc Natl Acad Sci U S A 2010 May 3;107(20):9072-7. Epub 2010 May 3.

Novartis Vaccines and Diagnostics, 53100 Siena, Italy.

Extraintestinal pathogenic Escherichia coli (ExPEC) are a common cause of disease in both mammals and birds. A vaccine to prevent such infections would be desirable given the increasing antibiotic resistance of these bacteria. We have determined the genome sequence of ExPEC IHE3034 (ST95) isolated from a case of neonatal meningitis and compared this to available genome sequences of other ExPEC strains and a few nonpathogenic E. coli. We found 19 genomic islands present in the genome of IHE3034, which are absent in the nonpathogenic E. coli isolates. By using subtractive reverse vaccinology we identified 230 antigens present in ExPEC but absent (or present with low similarity) in nonpathogenic strains. Nine antigens were protective in a mouse challenge model. Some of them were also present in other pathogenic non-ExPEC strains, suggesting that a broadly protective E. coli vaccine may be possible. The gene encoding the most protective antigen was detected in most of the E. coli isolates, highly conserved in sequence and found to be exported by a type II secretion system which seems to be nonfunctional in nonpathogenic strains.
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http://dx.doi.org/10.1073/pnas.0915077107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2889118PMC
May 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

Solution structure of the factor H-binding protein, a survival factor and protective antigen of Neisseria meningitidis.

J Biol Chem 2009 Apr 4;284(14):9022-6. Epub 2009 Feb 4.

Magnetic Resonance Center (CERM), F50019 Sesto Fiorentino, Italy.

Factor H-binding protein is a 27-kDa lipoprotein of Neisseria meningitidis discovered while screening the bacterial genome for vaccine candidates. In addition to being an important component of a vaccine against meningococcus in late stage of development, the protein is essential for pathogenesis because it allows the bacterium to survive and grow in human blood by binding the human complement factor H. We recently reported the solution structure of the C-terminal domain of factor H-binding protein, which contains the immunodominant epitopes. In the present study, we report the structure of the full-length molecule, determined by nuclear magnetic resonance spectroscopy. The protein is composed of two independent barrels connected by a short link. Mapping the residues recognized by monoclonal antibodies with bactericidal or factor H binding inhibition properties allowed us to predict the sites involved in the function of the protein. The structure therefore provides the basis for designing improved vaccine molecules.
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http://dx.doi.org/10.1074/jbc.C800214200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2666550PMC
April 2009

SR-A, MARCO and TLRs differentially recognise selected surface proteins from Neisseria meningitidis: an example of fine specificity in microbial ligand recognition by innate immune receptors.

J Innate Immun 2009 11;1(2):153-63. Epub 2008 Sep 11.

Sir William Dunn School of Pathology, University of Oxford, UK.

Macrophages express various classes of pattern recognition receptors involved in innate immune recognition of artificial, microbial and host-derived ligands. These include the scavenger receptors (SRs), which are important for phagocytosis, and the Toll-like receptors (TLRs) involved in microbe sensing. The class A macrophage scavenger receptor (SR-A) and macrophage receptor with a collagenous structure (MARCO) display similar domain structures and ligand-binding specificity, which has led to the assumption that these two receptors may be functionally redundant. In this study we show that SR-A and MARCO differentially recognise artificial polyanionic ligands as well as surface proteins from the pathogenic bacterium Neisseria meningitidis. We show that, while acetylated low-density lipoprotein (AcLDL) is a strong ligand for SR-A, it is not a ligand for MARCO. Of the neisserial proteins that were SR ligands, some were ligands for both receptors, while other proteins were only recognised by either SR-A or MARCO. We also analysed the potential of these ligands to act as TLR agonists and assessed the requirement for SR-A and MARCO in pro-inflammatory cytokine induction. SR ligation alone did not induce cytokine production; however, for proteins that were both SR and TLR ligands, the SRs were required for full activation of TLR pathways.
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http://dx.doi.org/10.1159/000155227DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7312862PMC
September 2010

Epitope mapping of a bactericidal monoclonal antibody against the factor H binding protein of Neisseria meningitidis.

J Mol Biol 2009 Feb 11;386(1):97-108. Epub 2008 Dec 11.

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

The factor H binding protein (fHbp) is a 27-kDa membrane-anchored lipoprotein of Neisseria meningitidis that allows the survival of the bacterium in human plasma; it is also a major component of a universal vaccine against meningococcus B. In this study, we used nuclear magnetic resonance spectroscopy, mutagenesis, and in silico modeling to map the epitope recognized by MAb502, a bactericidal monoclonal antibody elicited by fHbp. The data show that the antibody recognizes a conformational epitope within a well-defined area of the immunodominant C-terminal domain of the protein that is formed by two loops connecting different beta-strands of a beta-barrel and a short alpha-helix brought in spatial proximity by the protein folding. The identification of the protective epitopes of fHbp is an important factor for understanding the mechanism(s) of an effective immune response and provides valuable guidelines for designing variants of the protein able to induce broadly protective immunity.
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http://dx.doi.org/10.1016/j.jmb.2008.12.005DOI Listing
February 2009

Identification of Neisseria meningitidis nonlipopolysaccharide ligands for class A macrophage scavenger receptor by using a novel assay.

Infect Immun 2006 Sep;74(9):5191-9

Sir William Dunn School of Pathology, South Parks Road, Oxford OX1 3RE, United Kingdom.

Macrophages (Mphi) may play an important role in the pathogenesis of invasive meningococcal infection. Previously, we have shown that the class A Mphi scavenger receptor (SR-A) is a major nonopsonic receptor for Neisseria meningitidis on Mphi. SR-A contributes to host defense by binding proinflammatory polyanionic ligands such as lipopolysaccharide (LPS) and by the uptake and killing of live organisms. SR-A-deficient mouse Mphi display a substantial reduction in the number of meningococci ingested compared to wild-type Mphi, and SR-A is required for meningococcal phagocytosis but not for the release of tumor necrosis factor alpha. Although soluble lipid A and lipid(IV)A are reported as ligands for SR-A, we demonstrated that LPS and LPS expression were not essential for the uptake of whole meningococci. In the present study, we set out to discover protein ligand(s) for SR-A in N. meningitidis lysates and outer membrane vesicles. Using various microbial mutant strains, we determined that molecules comprising the membrane capsule and pili, as well as the abundant surface Opa proteins were not essential for SR-A recognition. We developed a binding assay to detect SR-A ligands and identified three candidate proteins expressed on intact organisms, namely, NMB1220, NMB0278, and NMB0667. Soluble forms of these ligands were shown to block the binding of meningococci to CHO cells stably transfected with SR-A. Furthermore, NMB1220 was endocytosed by SR-A on Mphi and prevented internalization of soluble acetylated low-density lipoprotein. Thus, we have identified novel, unmodified protein ligands for SR-A that are able to inhibit meningococcal interactions with macrophages in vitro.
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http://dx.doi.org/10.1128/IAI.00124-06DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1594824PMC
September 2006

A universal vaccine for serogroup B meningococcus.

Proc Natl Acad Sci U S A 2006 Jul 6;103(29):10834-9. Epub 2006 Jul 6.

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

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.
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http://dx.doi.org/10.1073/pnas.0603940103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2047628PMC
July 2006

Solution structure of the immunodominant domain of protective antigen GNA1870 of Neisseria meningitidis.

J Biol Chem 2006 Mar 31;281(11):7220-7. Epub 2005 Dec 31.

Centro Risonanze Magnetiche (CERM), University of Florence, Via L. Sacconi 6, 50019 Sesto Fiorentino, Italy.

GNA1870, a 28-kDa surface-exposed lipoprotein of Neisseria meningitidis recently discovered by reverse vaccinology, is one of the most potent antigens of Meningococcus and a promising candidate for a universal vaccine against a devastating disease. Previous studies of epitope mapping and genetic characterization identified residues critical for bactericidal response within the C-terminal domain of the molecule. To elucidate the conformation of protective epitopes, we used NMR spectroscopy to obtain the solution structure of the immunodominant 18-kDa C-terminal portion of GNA1870. The structure consists of an eight-stranded antiparallel beta-barrel overlaid by a short alpha-helix with an unstructured N-terminal end. Residues previously shown to be important for antibody recognition were mapped on loops facing the same ridge of the molecule. The sequence similarity of GNA1870 with members of the bacterial transferrin receptor family allows one to predict the folding of this class of well known bacterial antigens, providing the basis for the rational engineering of high affinity B cell epitopes.
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http://dx.doi.org/10.1074/jbc.M508595200DOI Listing
March 2006

Ng-MIP, a surface-exposed lipoprotein of Neisseria gonorrhoeae, has a peptidyl-prolyl cis/trans isomerase (PPIase) activity and is involved in persistence in macrophages.

Mol Microbiol 2005 Nov;58(3):669-81

IRIS, Chiron S.r.l., Via Fiorentina, 1, 53100 Siena, Italy.

Macrophage infectivity potentiators (MIPs) are a family of surface-exposed virulence factors of intracellular microorganisms such as Legionella, Chlamydia and Trypanosoma. These proteins display peptidyl-prolyl cis/trans isomerase (PPIase) activity that is inhibited by immunosuppressants FK506 and rapamycin. Here we describe the identification and characterization in Neisseria gonorrhoeae of Ng-MIP, a surface-exposed lipoprotein with high homology to MIPs. The protein is an homodimer with rapamycin-inhibited PPIase activity confirming that it is a functional member of the MIP family. A knock-out strain, generated by deletion of the mip gene in N. gonorrhoeae F62 strain, was evaluated for its role in infection of mouse and human macrophages. We show that Ng-MIP promotes the intracellular survival of N. gonorrhoeae in macrophages, highlighting a possible role of this protein in promoting the persistence of gonococcal infection.
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http://dx.doi.org/10.1111/j.1365-2958.2005.04859.xDOI Listing
November 2005