Publications by authors named "Werner Pansegrau"

20 Publications

  • Page 1 of 1

Stability of Outer Membrane Vesicles-Based Vaccines, Identifying the Most Appropriate Methods to Detect Changes in Vaccine Potency.

Vaccines (Basel) 2021 Mar 6;9(3). Epub 2021 Mar 6.

GSK Vaccines Institute for Global Health (GVGH) S.r.l., Via Fiorentina 1, 53100 Siena, Italy.

Ensuring the stability of vaccines is crucial to successfully performing global immunization programs. Outer Membrane Vesicles (OMV) are receiving great attention as vaccine platforms. OMV are complex molecules and few data have been collected so far on their stability. OMV produced by bacteria, genetically modified to increase their spontaneous release, simplifying their production, are also known as Generalized Modules for Membrane Antigens (GMMA). We have performed accelerated stability studies on GMMA from different pathogens and verified the ability of physico-chemical and immunological methods to detect possible changes. High-temperature conditions (100 °C for 40 min) did not affect GMMA stability and immunogenicity in mice, in contrast to the effect of milder temperatures for a longer period of time (37 °C or 50 °C for 4 weeks). We identified critical quality attributes to monitor during stability assessment that could impact vaccine efficacy. In particular, specific recognition of antigens by monoclonal antibodies through competitive ELISA assays may replace in vivo tests for the potency assessment of GMMA-based vaccines.
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http://dx.doi.org/10.3390/vaccines9030229DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7998687PMC
March 2021

Epitope and Paratope Mapping by HDX-MS Combined with SPR Elucidates the Difference in Bactericidal Activity of Two Anti-NadA Monoclonal Antibodies.

J Am Soc Mass Spectrom 2021 Jul 8;32(7):1575-1582. Epub 2021 Mar 8.

Protein Analysis Group, Department of Pharmacy, University of Copenhagen, 2100 Copenhagen O, Denmark.

Characterization of antigen-antibody interactions is crucial for understanding antibody-mediated protection against pathogens, biopharmaceutical development, as well as evaluation of the immune response post vaccination. Bexsero is a multicomponent vaccine against serogroup B in which one of the key vaccine antigens is Neisserial adhesin A (NadA), a trimeric coiled-coil protein. Two NadA-specific monoclonal antibodies (mAbs) isolated from Bexsero-vaccinated individuals have been shown to have similar binding affinity and appear to recognize a similar antigen region, yet only one of the mAbs is bactericidal. In this study, we use hydrogen/deuterium exchange mass spectrometry (HDX-MS) to perform an in-depth study of the interaction of the two mAbs with NadA antigen using a combined epitope and paratope mapping strategy. In addition, we use surface plasmon resonance (SPR) to investigate the stoichiometry of the binding of the two mAbs to NadA. While epitope mapping only identifies a clear binding impact of one of the mAbs on NadA, the paratope mapping analyses shows that both mAbs are binding to NadA through several complementarity determining regions spanning both heavy and light chains. Our results highlight the advantage of combined epitope and paratope mapping HDX-MS experiments and supporting biochemical experiments to characterize antigen-antibody interactions. Through this combined approach, we provide a rationale for how the binding stoichiometry of the two mAbs to the trimeric NadA antigen can explain the difference in bactericidal activity of the two mAbs.
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http://dx.doi.org/10.1021/jasms.0c00431DOI Listing
July 2021

4CMenB vaccine induces elite cross-protective human antibodies that compete with human factor H for binding to meningococcal fHbp.

PLoS Pathog 2020 10 2;16(10):e1008882. Epub 2020 Oct 2.

GSK, Siena, Italy.

Neisseria meningitidis serogroup B (MenB) is the leading cause of meningococcal meningitis and sepsis in industrialized countries, with the highest incidence in infants and adolescents. Two recombinant protein vaccines that protect against MenB are now available (i.e. 4CMenB and MenB-fHbp). Both vaccines contain the Factor H Binding Protein (fHbp) antigen, which can bind the Human Factor H (fH), the main negative regulator of the alternative complement pathway, thus enabling bacterial survival in the blood. fHbp is present in meningococcal strains as three main variants which are immunologically distinct. Here we sought to obtain detailed information about the epitopes targeted by anti-fHbp antibodies induced by immunization with the 4CMenB multicomponent vaccine. Thirteen anti-fHbp human monoclonal antibodies (mAbs) were identified in a library of over 100 antibody fragments (Fabs) obtained from three healthy adult volunteers immunized with 4CMenB. Herein, the key cross-reactive mAbs were further characterized for antigen binding affinity, complement-mediated serum bactericidal activity (SBA) and the ability to inhibit binding of fH to live bacteria. For the first time, we identified a subset of anti-fHbp mAbs able to elicit human SBA against strains with all three variants and able to compete with human fH for fHbp binding. We present the crystal structure of fHbp v1.1 complexed with human antibody 4B3. The structure, combined with mutagenesis and binding studies, revealed the critical cross-reactive epitope. The structure also provided the molecular basis of competition for fH binding. These data suggest that the fH binding site on fHbp v1.1 can be accessible to the human immune system upon immunization, enabling elicitation of human mAbs broadly protective against MenB. The novel structural, biochemical and functional data are of great significance because the human vaccine-elicited mAbs are the first reported to inhibit the binding of fH to fHbp, and are bactericidal with human complement. Our studies provide molecular insights into the human immune response to the 4CMenB meningococcal vaccine and fuel the rationale for combined structural, immunological and functional studies when seeking deeper understanding of the mechanisms of action of human vaccines.
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http://dx.doi.org/10.1371/journal.ppat.1008882DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7556464PMC
October 2020

Synergic complement-mediated bactericidal activity of monoclonal antibodies with distinct specificity.

FASEB J 2020 08 17;34(8):10329-10341. Epub 2020 Jun 17.

GSK, Siena, Italy.

The classical complement pathway is triggered when antigen-bound immunoglobulins bind to C1q through their Fc region. While C1q binds to a single Fc with low affinity, a higher avidity stable binding of two or more of C1q globular heads initiates the downstream reactions of the complement cascade ultimately resulting in bacteriolysis. Synergistic bactericidal activity has been demonstrated when monoclonal antibodies recognize nonoverlapping epitopes of the same antigen. The aim of the present work was to investigate the synergistic effect between antibodies directed toward different antigens. To this purpose, we investigated the bactericidal activity induced by combinations of monoclonal antibodies (mAbs) raised against factor H-binding protein (fHbp) and Neisserial Heparin-Binding Antigen (NHBA), two major antigens included in Bexsero, the vaccine against Meningococcus B, for prevention from this devastating disease in infants and adolescents. Collectively, our results show that mAbs recognizing different antigens can synergistically activate complement even when each single Mab is not bactericidal, reinforcing the evidence that cooperative immunity induced by antigen combinations can represent a remarkable added value of multicomponent vaccines. Our study also shows that the synergistic effect of antibodies is modulated by the nature of the respective epitopes, as well as by the antigen density on the bacterial cell surface.
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http://dx.doi.org/10.1096/fj.201902795RDOI Listing
August 2020

A LONELY GUY protein of Bordetella pertussis with unique features is related to oxidative stress.

Sci Rep 2019 11 19;9(1):17016. Epub 2019 Nov 19.

School of Biosciences and Veterinary Medicine, University of Camerino, via Gentile III da Varano, 62032, Camerino, Italy.

The Gram-negative bacterium B. pertussis is the causative agent of whooping cough. This infection is re-emerging and new features related to Bordetella pathogenesis and microbiology could be relevant to defeat it. Therefore, we focused our attention on BP1253, a predicted exported protein from B. pertussis erroneously classified as lysine decarboxylase. We showed that BP1253 shares the highly conserved motif PGGxGTxxE and the key catalytic amino-acid residues with newly structurally characterized "LONELY GUY" (LOG) proteins. Biochemical studies have confirmed that this protein functions as a cytokinin-activating enzyme since it cleaves the N-glycosidic linkage between the base and the ribose, leading to the formation of free bases, which are the active form of plant hormones called cytokinins. Remarkably, BP1253 selectively binds monophosphate nucleotides such as AMP, GMP and CMP, showing a wider variety in binding capacity compared to other LOGs. Cytokinin production studies performed with B. pertussis have revealed 6-O-methylguanine to be the physiological product of BP1253 in agreement with the higher activity of the enzyme towards GMP. 6-O-methylguanine is likely to be responsible for the increased sensitivity of B. pertussis to oxidative stress. Although BP1253 has a primary sequence resembling the hexameric type-II LOGs, the dimeric state and the presence of specific amino-acids suggests that BP1253 can be classified as a novel type-II LOG. The discovery of a LOG along with its product 6-O-methylguanine in the human pathogen B. pertussis may lead to the discovery of unexplored functions of LOGs, broadening their role beyond plants.
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http://dx.doi.org/10.1038/s41598-019-53171-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6864091PMC
November 2019

Structural basis for cooperativity of human monoclonal antibodies to meningococcal factor H-binding protein.

Commun Biol 2019 26;2:241. Epub 2019 Jun 26.

GSK Vaccines Srl, 53100 Siena, Italy.

Monoclonal antibody (mAb) cooperativity is a phenomenon triggered when mAbs couples promote increased bactericidal killing compared to individual partners. Cooperativity has been deeply investigated among mAbs elicited by factor H-binding protein (fHbp), a surface-exposed lipoprotein and one of the key antigens included in both serogroup B meningococcus vaccine Bexsero and Trumenba. Here we report the structural and functional characterization of two cooperative mAbs pairs isolated from Bexsero vaccines. The 3D electron microscopy structures of the human mAb-fHbp-mAb cooperative complexes indicate that the angle formed between the antigen binding fragments (fAbs) assume regular angle and that fHbp is able to bind simultaneously and stably the cooperative mAbs pairs and human factor H (fH) in vitro. These findings shed light on molecular basis of the antibody-based mechanism of protection driven by simultaneous recognition of the different epitopes of the fHbp and underline that cooperativity is crucial in vaccine efficacy.
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http://dx.doi.org/10.1038/s42003-019-0493-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6595007PMC
April 2020

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

Pilus Assembly in Gram-Positive Bacteria.

Curr Top Microbiol Immunol 2017 ;404:203-233

GSK Vaccines, Siena, Italy.

Pili of Gram-positive bacteria are unique structures on the bacterial surface, assembled from covalently linked polypeptide subunits. Pilus assembly proceeds by transpeptidation reactions catalyzed by sortases, followed by covalent anchoring of the filament in the peptidoglycan layer. Another distinctive property is the presence of intramolecular isopeptide bonds, conferring extraordinary chemical and mechanical stability to these elongated structures. Besides their function in cell adhesion and biofilm formation, this section discusses possible application of pilus constituents as vaccine components against Gram-positive pathogens.
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http://dx.doi.org/10.1007/82_2015_5016DOI Listing
August 2017

Identification of a Monoclonal Antibody Against Pneumococcal Pilus 1 Ancillary Protein Impairing Bacterial Adhesion to Human Epithelial Cells.

J Infect Dis 2016 Feb 23;213(4):516-22. Epub 2015 Sep 23.

GSK Vaccines, Siena.

The adhesion of Streptococcus pneumoniae is a key step during colonization of human respiratory tract mucosae. Here we demonstrate that pneumococcal type I pilus significantly increases the adhesiveness of poorly adhering highly capsulated strains in vitro. Interestingly, preincubation of bacteria with antibodies against the major pilus backbone subunit (RrgB) or the adhesin component (RrgA) impaired pneumococcal association to human epithelial cells. Screening for anti-RrgA monoclonal antibodies specifically affecting the adhesive capacity of S. pneumoniae led to the identification of the monoclonal 11B9/61 antibody, which greatly reduced pilus-dependent cell contact. Proteomic-based epitope mapping of 11B9/61 monoclonal antibody revealed a well-exposed epitope on the D2 domain of RrgA as the target of this functional antibody. The data presented here confirm the importance of pilus I for S. pneumoniae pathogenesis and the potential use of antipilus antibodies to prevent bacterial colonization.
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http://dx.doi.org/10.1093/infdis/jiv461DOI Listing
February 2016

Defined conjugation of glycans to the lysines of CRM197 guided by their reactivity mapping.

Chembiochem 2014 Apr 11;15(6):836-43. Epub 2014 Mar 11.

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

Systematic characterisation of the reactivity of the lysine moieties in CRM197 towards N-hydroxysuccinimide linkers bearing alkynes or azides is described. This involves two-step conjugation of various glycans to CRM197 by click chemistry in a well-defined manner. By semiquantitative LC-MS/MS analysis of proteolytic digests of the conjugates formed, the reactivity of lysine residues in the protein was mapped and ranked. Computational analysis of the solvent accessibility of each lysine residue (based on the CRM197 crystal structure) established a correlation between reactivity and surface exposure. By this approach, conjugation involving lysine residues (normally a random process) can be controlled. It enables the preparation of lysine-mediated glycoconjugates with improved batch-to-batch reproducibility, thereby producing neo-glycoconjugates with more-consistent biological activity.
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http://dx.doi.org/10.1002/cbic.201300785DOI Listing
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

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

Structural and functional characterization of the Streptococcus pneumoniae RrgB pilus backbone D1 domain.

J Biol Chem 2011 Apr 2;286(16):14588-97. Epub 2011 Mar 2.

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

Streptococcus pneumoniae expresses on its surface adhesive pili, involved in bacterial attachment to epithelial cells and virulence. The pneumococcal pilus is composed of three proteins, RrgA, RrgB, and RrgC, each stabilized by intramolecular isopeptide bonds and covalently polymerized by means of intermolecular isopeptide bonds to form an extended fiber. RrgB is the pilus scaffold subunit and is protective in vivo in mouse models of sepsis and pneumonia, thus representing a potential vaccine candidate. The crystal structure of a major RrgB C-terminal portion featured an organization into three independently folded protein domains (D2-D4), whereas the N-terminal D1 domain (D1) remained unsolved. We have tested the four single recombinant RrgB domains in active and passive immunization studies and show that D1 is the most effective, providing a level of protection comparable with that of the full-length protein. To elucidate the structural features of D1, we solved the solution structure of the recombinant domain by NMR spectroscopy. The spectra analysis revealed that D1 has many flexible regions, does not contain any intramolecular isopeptide bond, and shares with the other domains an Ig-like fold. In addition, we demonstrated, by site-directed mutagenesis and complementation in S. pneumoniae, that the D1 domain contains the Lys residue (Lys-183) involved in the formation of the intermolecular isopeptide bonds and pilus polymerization. Finally, we present a model of the RrgB protein architecture along with the mapping of two surface-exposed linear epitopes recognized by protective antisera.
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http://dx.doi.org/10.1074/jbc.M110.202739DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3077656PMC
April 2011

Adhesion determinants of the Streptococcus species.

Microb Biotechnol 2010 Jul 31;3(4):370-88. Epub 2009 Jul 31.

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

Streptococci are clinically important Gram-positive bacteria that are capable to cause a wide variety of diseases in humans and animals. Phylogenetic analyses based on 16S rRNA sequences of the streptococcal species reveal a clustering pattern, reflecting, with a few exceptions, their pathogenic potential and ecological preferences. Microbial adhesion to host tissues is the initial critical event in the pathogenesis of most infections. Streptococci use multiple adhesins to attach to the epithelium, and their expression is regulated in response to environmental and growth conditions. Bacterial adhesins recognize and bind cell surface molecules and extracellular matrix components through specific domains that for certain adhesin families have been well defined and found conserved across the streptococcal species. In this review, we present the different streptococcal adhesin families categorized on the basis of their adhesive properties and structural characteristics, and, when available, we focus the attention on conserved functional domains.
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http://dx.doi.org/10.1111/j.1751-7915.2009.00138.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3815805PMC
July 2010

The two variants of the Streptococcus pneumoniae pilus 1 RrgA adhesin retain the same function and elicit cross-protection in vivo.

Infect Immun 2010 Dec 7;78(12):5033-42. Epub 2010 Sep 7.

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

Thirty percent of Streptococcus pneumoniae isolates contain pilus islet 1, coding for a pilus composed of the backbone subunit RrgB and two ancillary proteins, RrgA and RrgC. RrgA is the major determinant of in vitro adhesion associated with pilus 1, is protective in vivo in mouse models, and exists in two variants (clades I and II). Mapping of the sequence variability onto the RrgA structure predicted from X-ray data showed that the diversity was restricted to the "head" of the protein, which contains the putative binding domains, whereas the elongated "stalk" was mostly conserved. To investigate whether this variability could influence the adhesive capacity of RrgA and to map the regions important for binding, two full-length protein variants and three recombinant RrgA portions were tested for adhesion to lung epithelial cells and to purified extracellular matrix (ECM) components. The two RrgA variants displayed similar binding abilities, whereas none of the recombinant fragments adhered at levels comparable to those of the full-length protein, suggesting that proper folding and structural arrangement are crucial to retain protein functionality. Furthermore, the two RrgA variants were shown to be cross-reactive in vitro and cross-protective in vivo in a murine model of passive immunization. Taken together, these data indicate that the region implicated in adhesion and the functional epitopes responsible for the protective ability of RrgA may be conserved and that the considerable level of variation found within the "head" domain of RrgA may have been generated by immunologic pressure without impairing the functional integrity of the pilus.
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http://dx.doi.org/10.1128/IAI.00601-10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2981310PMC
December 2010

Sortase A confers protection against Streptococcus pneumoniae in mice.

Infect Immun 2009 Jul 11;77(7):2957-61. Epub 2009 May 11.

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

Streptococcus pneumoniae sortase A (SrtA) is a transpeptidase that is highly conserved among pneumococcal strains, whose involvement in adhesion/colonization has been reported. We found that intraperitoneal immunization with recombinant SrtA conferred to mice protection against S. pneumoniae intraperitoneal challenge and that the passive transfer of immune serum before intraperitoneal challenge was also protective. Moreover, by using the intranasal challenge model, we observed a significant reduction of bacteremia when mice were intraperitoneally immunized with SrtA, while a moderate decrease of lung infection was achieved by intranasal immunization, even though no influence on nasopharynx colonization was seen. Taken together, our results suggest that SrtA is a good candidate for inclusion in a multicomponent, protein-based, pneumococcal vaccine.
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http://dx.doi.org/10.1128/IAI.01516-08DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2708569PMC
July 2009

A second pilus type in Streptococcus pneumoniae is prevalent in emerging serotypes and mediates adhesion to host cells.

J Bacteriol 2008 Aug 30;190(15):5480-92. Epub 2008 May 30.

Department of Cellular Microbiology and Bioinformatics, Novartis Vaccines, University of Siena, Siena 53100, Italy.

Analysis of publicly available genomes of Streptococcus pneumoniae has led to the identification of a new genomic element containing genes typical of gram-positive pilus islets (PIs). Here, we demonstrate that this genomic region, herein referred to as PI-2 (consisting of pitA, sipA, pitB, srtG1, and srtG2) codes for a second functional pilus in pneumococcus. Polymerization of the PI-2 pilus requires the backbone protein PitB as well as the sortase SrtG1 and the signal peptidase-like protein SipA. Presence of PI-2 correlates with the genotype as defined by multilocus sequence typing and clonal complex (CC). The PI-2-positive CCs are associated with serotypes 1, 2, 7F, 19A, and 19F, considered to be emerging serotypes in both industrialized and developing countries. Interestingly, strains belonging to CC271 (where sequence type 271 is the predicted founder of the CC) contain both PI-1 and PI-2, as revealed by genome analyses. In these strains both pili are surface exposed and independently assembled. Furthermore, in vitro experiments provide evidence that the pilus encoded by PI-2 of S. pneumoniae is involved in adherence. Thus, pneumococci encode at least two types of pili that play a role in the initial host cell contact to the respiratory tract and are potential antigens for inclusion in a new generation of pneumococcal vaccines.
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http://dx.doi.org/10.1128/JB.00384-08DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2493256PMC
August 2008

Streptococcus pneumoniae pilus subunits protect mice against lethal challenge.

Infect Immun 2007 Feb 4;75(2):1059-62. Epub 2006 Dec 4.

Novartis Vaccines and Diagnostics s.r.l., Research Center, Via Fiorentina 1, I-53100 Siena, Italy.

Streptococcus pneumoniae is a major public health threat worldwide. The recent discovery that this pathogen possesses pili led us to investigate their protective abilities in a mouse model of intraperitoneal infection. Both active and passive immunization with recombinant pilus subunits afforded protection against lethal challenge with the S. pneumoniae serotype 4 strain TIGR4.
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http://dx.doi.org/10.1128/IAI.01400-06DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1828493PMC
February 2007

Inhibitors of Helicobacter pylori ATPase Cagalpha block CagA transport and cag virulence.

Microbiology (Reading) 2006 Oct;152(Pt 10):2919-2930

Cellular Microbiology and Bioinformatics Unit, Immunological Research Institute Siena (IRIS), Novartis Vaccines S.r.l., Via Fiorentina 1, I-53100 Siena, Italy.

With the steadily increasing occurrence of antibiotic resistance in bacteria, there is a great need for new antibacterial compounds. The approach described here involves targeting virulence-related bacterial type IV secretion systems (TFSSs) with small-molecule inhibitors. The cag TFSS of Helicobacter pylori was chosen as a model, and novel inhibitors directed against the cag VirB11-type ATPase Cagalpha were identified. The cag genes encode proteins that are components of a contact-dependent secretion system used by the bacterium to translocate the effector molecule CagA into host cells. Translocated CagA is associated with severe gastritis, and carcinoma. Furthermore, functional TFSSs and immunodominant CagA play a role in interleukin (IL)-8 induction, which is an important factor for chronic inflammation. Inhibitors of Cagalpha were identified by high-throughput screening of chemical libraries that comprised 524 400 small molecules. The ATPase activity of Cagalpha was inhibited by the selected compounds in an in vitro enzymic assay using the purified enzyme. The most active compound, CHIR-1, reduced TFSS function to an extent that cellular effects on AGS cells mediated by CagA were virtually undetectable, while reduced levels of IL-8 induction were observed. Gastric colonization by CHIR-1-pre-treated bacteria was found to be impaired in a dose-dependent manner using a mouse model of infection. Small-molecule Cagalpha inhibitors, the first described inhibitors of a TFSS, are potential candidates for the development of new antibacterial compounds that may lead to alternative medical treatments. The compounds are expected to impose weak selective pressure, since they target virulence functions. Moreover, the targeted virulence protein is conserved in a variety of bacterial pathogens. Additionally, TFSS inhibitors are potent tools to study the biology of TFSSs.
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http://dx.doi.org/10.1099/mic.0.28984-0DOI Listing
October 2006