Publications by authors named "Maria Scarselli"

78 Publications

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

Vaccines to Overcome Antibiotic Resistance: The Challenge of Burkholderia cenocepacia.

Trends Microbiol 2020 04 10;28(4):315-326. Epub 2020 Jan 10.

Department of Biology and Biotechnology 'Lazzaro Spallanzani', University of Pavia, Pavia, Italy. Electronic address:

Cystic fibrosis (CF) patients are at particular risk of infection by microorganisms that are resistant to several antibiotics. About 3% of CF patients are colonized by Burkholderia cenocepacia, and this represents a major threat because of its intrinsic high level of drug resistance and the lack of a safe and effective treatment protocol. The development of anti-Burkholderia vaccines is a valuable and complementary approach, but only a few studies have been reported to date. In this review we discuss recent advances in the vaccine field and how new technologies, including structural reverse vaccinology, could drive the design of an effective vaccine against B. cenocepacia for use in preventive and therapeutic applications.
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http://dx.doi.org/10.1016/j.tim.2019.12.005DOI Listing
April 2020

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

Dual role of the colonization factor CD2831 in Clostridium difficile pathogenesis.

Sci Rep 2019 04 3;9(1):5554. Epub 2019 Apr 3.

Glaxo Smith Kline Vaccines, Via Fiorentina 1, 53100, Siena, Italy.

Clostridium difficile is a Gram-positive, anaerobic bacterium and the leading cause of antibiotic-associated diarrhea and pseudomembranous colitis. C. difficile modulates its transition from a motile to a sessile lifestyle through a mechanism of riboswitches regulated by cyclic diguanosine monophosphate (c-di-GMP). Previously described as a sortase substrate positively regulated by c-di-GMP, CD2831 was predicted to be a collagen-binding protein and thus potentially involved in sessility. By overexpressing CD2831 in C. difficile and heterologously expressing it on the surface of Lactococcus lactis, here we further demonstrated that CD2831 is a collagen-binding protein, able to bind to immobilized collagen types I, III and V as well as native collagen produced by human fibroblasts. We also observed that the overexpression of CD2831 raises the ability to form biofilm on abiotic surface in both C. difficile and L. lactis. Notably, we showed that CD2831 binds to the collagen-like domain of the human complement component C1q, suggesting a role in preventing complement cascade activation via the classical pathway. This functional characterization places CD2831 in the Microbial Surface Components Recognizing Adhesive Matrix Molecule (MSCRAMMs) family, a class of virulence factors with a dual role in adhesion to collagen-rich tissues and in host immune evasion by binding to human complement components.
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http://dx.doi.org/10.1038/s41598-019-42000-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6447587PMC
April 2019

Genome-Based Approach Delivers Vaccine Candidates Against .

Front Immunol 2018 9;9:3021. Epub 2019 Jan 9.

Infection and Cystic Fibrosis Unit, Division of Immunology, Transplantation and Infectious Diseases, IRCCS San Raffaele Scientific Institute, Milan, Italy.

High incidence, severity and increasing antibiotic resistance characterize infections, highlighting the need for new therapeutic options. Vaccination strategies to prevent or limit infections represent a rational approach to positively impact the clinical outcome of risk patients; nevertheless this bacterium remains a challenging vaccine target. To identify novel vaccine candidates, we started from the genome sequence analysis of the reference strain PAO1 exploring the reverse vaccinology approach integrated with additional bioinformatic tools. The bioinformatic approaches resulted in the selection of 52 potential antigens. These vaccine candidates were conserved in genomes from different origin and among strains isolated longitudinally from cystic fibrosis patients. To assess the immune-protection of single or antigens combination against infection, a vaccination protocol was established in murine model of acute respiratory infection. Combinations of selected candidates, rather than single antigens, effectively controlled infection in the model of murine pneumonia. Five combinations were capable of significantly increase survival rate among challenged mice and all included PA5340, a hypothetical protein exclusively present in . PA5340 combined with PA3526-MotY gave the maximum protection. Both proteins were surface exposed by immunofluorescence and triggered a specific immune response. Combination of these two protein antigens could represent a potential vaccine to prevent infection.
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http://dx.doi.org/10.3389/fimmu.2018.03021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6334337PMC
October 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

Native State Organization of Outer Membrane Porins Unraveled by HDx-MS.

J Proteome Res 2018 05 17;17(5):1794-1800. Epub 2018 Apr 17.

GSK , Via Fiorentina 1 , 53100 Siena , Italy.

Hydrogen-deuterium exchange (HDx) associated with mass spectrometry (MS) is emerging as a powerful tool to provide conformational information about membrane proteins. Unfortunately, as for X-ray diffraction and NMR, HDx performed on reconstituted in vitro systems might not always reflect the in vivo environment. Outer-membrane vesicles naturally released by Escherichia coli were used to carry out analysis of native OmpF through HDx-MS. A new protocol compatible with HDx analysis that avoids hindrance from the lipid contents was setup. The extent of deuterium incorporation was in good agreement with the X-ray diffraction data of OmpF as the buried β-barrels incorporated a low amount of deuterium, whereas the internal loop L3 and the external loops incorporated a higher amount of deuterium. Moreover, the kinetics of incorporation clearly highlights that peptides segregate well in two distinct groups based exclusively on a trimeric organization of OmpF in the membrane: peptides presenting fast kinetics of labeling are facing the complex surrounding environment, whereas those presenting slow kinetics are located in the buried core of the trimer. The data show that HDx-MS applied to a complex biological system is able to reveal solvent accessibility and spatial arrangement of an integral outer-membrane protein complex.
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http://dx.doi.org/10.1021/acs.jproteome.7b00830DOI Listing
May 2018

CagL from Helicobacter pylori has ADP-ribosylation activity and exerts partial protective efficacy in mice.

Arch Biochem Biophys 2017 12 31;635:102-109. Epub 2017 Oct 31.

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

Mono ADP-ribosyltransferases are a class of functionally conserved enzymes present in prokaryotic and eukaryotic organisms. In prokaryotes, mono ADP-ribose transfer enzymes often represent a family of exotoxins that display activity in a variety of bacteria responsible for causing disease in plants and animals. A bioinformatic approach has allowed us to identify that CagL gene from some Helicobacter pylori strains shares a sequence pattern with ADP-ribosylating toxins of the CT-group. In this manuscript we show that recombinant CagL from Shi470 is catalytically active showing ADP-ribosyltransferase, NAD-glycohydrolase, and auto-ADP-ribosylation activities. This is the first time that a catalytically active member of the ADP-ribosyltransferase family is identified in Helicobacter pylori. This observation may lead to the discovery of novel functions exerted by CagL in the pathogenesis of Helicobacter pylori. Indeed, we have shown that vaccination with CagL has protective efficacy in mice indicating that CagL may be considered as potential component of a Helicobacter pylori vaccine.
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http://dx.doi.org/10.1016/j.abb.2017.10.019DOI Listing
December 2017

Neisseria meningitidis factor H-binding protein bound to monoclonal antibody JAR5: implications for antibody synergy.

Biochem J 2016 Dec 26;473(24):4699-4713. Epub 2016 Oct 26.

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

Factor H-binding protein (fHbp) is an important antigen of Neisseria meningitidis that is capable of eliciting a robust protective immune response in humans. Previous studies on the interactions of fHbp with antibodies revealed that some anti-fHbp monoclonal antibodies that are unable to trigger complement-mediated bacterial killing in vitro are highly co-operative and become bactericidal if used in combination. Several factors have been shown to influence such co-operativity, including IgG subclass and antigen density. To investigate the structural basis of the anti-fHbp antibody synergy, we determined the crystal structure of the complex between fHbp and the Fab (fragment antigen-binding) fragment of JAR5, a specific anti-fHbp murine monoclonal antibody known to be highly co-operative with other monoclonal antibodies. We show that JAR5 is highly synergic with monoclonal antibody (mAb) 12C1, whose structure in complex with fHbp has been previously solved. Structural analyses of the epitopes recognized by JAR5 and 12C1, and computational modeling of full-length IgG mAbs of JAR5 and 12C1 bound to the same fHbp molecule, provide insights into the spatial orientation of Fc (fragment crystallizable) regions and into the possible implications for the susceptibility of meningococci to complement-mediated killing.
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http://dx.doi.org/10.1042/BCJ20160806DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6398935PMC
December 2016

Auto-Assembling Detoxified Staphylococcus aureus Alpha-Hemolysin Mimicking the Wild-Type Cytolytic Toxin.

Clin Vaccine Immunol 2016 06 6;23(6):442-50. Epub 2016 Jun 6.

GSK Vaccines, Research Center, Siena, Italy

Staphylococcus aureus alpha-hemolysin (Hla) assembles into heptameric pores on the host cell membrane, causing lysis, apoptosis, and junction disruption. Herein, we present the design of a newly engineered S. aureus alpha-toxin, HlaPSGS, which lacks the predicted membrane-spanning stem domain. This protein is able to form heptamers in aqueous solution in the absence of lipophilic substrata, and its structure, obtained by transmission electron microscopy and single-particle reconstruction analysis, resembles the cap of the wild-type cytolytic Hla pore. HlaPSGS was found to be impaired in binding to host cells and to its receptor ADAM10 and to lack hemolytic and cytotoxic activity. Immunological studies using human sera as well as sera from mice convalescent from S. aureus infection suggested that the heptameric conformation of HlaPSGS mimics epitopes exposed by the cytolytic Hla pore during infection. Finally, immunization with this newly engineered Hla generated high protective immunity against staphylococcal infection in mice. Overall, this study provides unprecedented data on the natural immune response against Hla and suggests that the heptameric HlaPSGS is a highly valuable vaccine candidate against S. aureus.
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http://dx.doi.org/10.1128/CVI.00091-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4895005PMC
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

Structural characterization of zinc-bound Zmp1, a zinc-dependent metalloprotease secreted by Clostridium difficile.

J Biol Inorg Chem 2016 Apr 28;21(2):185-96. Epub 2015 Dec 28.

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

Proteases are commonly secreted by microorganisms. In some pathogens, they can play a series of functional roles during infection, including maturation of cell surface or extracellular virulence factors, interference with host cell signaling, massive host tissue destruction, and dissolution of infection-limiting clots through degradation of the host proteins devoted to the coagulation cascade. We previously reported the identification and characterization of Zmp1, a zinc-dependent metalloprotease secreted by Clostridium difficile, demonstrated that Zmp1 is able to degrade fibrinogen in vitro, and identified two residues necessary to the catalytic activity. In the present work, we solved the solution structure of Zmp1 by Nuclear Magnetic Resonance (NMR) and compared it with the recently solved X-ray structures of substrate-bound and substrate-free Zmp1, highlighting similarities and differences. We also combined the structural characterization to biochemical assays and site-directed mutagenesis, to provide new insights into the catalytic site and on the residues responsible for substrate specificity. The Zmp1 structure showed similarity to the catalytic domain of Anthrax Lethal Factor of Bacillus anthracis. Analogies and differences in the catalytic and in the substrate-binding sites of the two proteins are discussed.
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http://dx.doi.org/10.1007/s00775-015-1319-6DOI Listing
April 2016

Molecular Engineering of Ghfp, the Gonococcal Orthologue of Neisseria meningitidis Factor H Binding Protein.

Clin Vaccine Immunol 2015 Jul 6;22(7):769-77. Epub 2015 May 6.

Novartis Vaccines Srl, a GSK Company, Siena, Italy

Knowledge of the sequences and structures of proteins produced by microbial pathogens is continuously increasing. Besides offering the possibility of unraveling the mechanisms of pathogenesis at the molecular level, structural information provides new tools for vaccine development, such as the opportunity to improve viral and bacterial vaccine candidates by rational design. Structure-based rational design of antigens can optimize the epitope repertoire in terms of accessibility, stability, and variability. In the present study, we used epitope mapping information on the well-characterized antigen of Neisseria meningitidis factor H binding protein (fHbp) to engineer its gonococcal homologue, Ghfp. Meningococcal fHbp is typically classified in three distinct antigenic variants. We introduced epitopes of fHbp variant 1 onto the surface of Ghfp, which is naturally able to protect against meningococcal strains expressing fHbp of variants 2 and 3. Heterologous epitopes were successfully transplanted, as engineered Ghfp induced functional antibodies against all three fHbp variants. These results confirm that structural vaccinology represents a successful strategy for modulating immune responses, and it is a powerful tool for investigating the extension and localization of immunodominant epitopes.
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http://dx.doi.org/10.1128/CVI.00794-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4478525PMC
July 2015

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

Neisseria meningitidis factor H-binding protein fHbp: a key virulence factor and vaccine antigen.

Expert Rev Vaccines 2015 Jun 23;14(6):841-59. Epub 2015 Feb 23.

Institute for Glycomics, Griffith University, Southport, Queensland, 4215, Australia.

Neisseria meningitidis is a leading cause of meningitis and sepsis worldwide. The first broad-spectrum multicomponent vaccine against serogroup B meningococcus (MenB), 4CMenB (Bexsero(®)), was approved by the EMA in 2013, for prevention of MenB disease in all age groups, and by the US FDA in January 2015 for use in adolescents. A second protein-based MenB vaccine has also been approved in the USA for adolescents (rLP2086, Trumenba(®)). Both vaccines contain the lipoprotein factor H-binding protein (fHbp). Preclinical studies demonstrated that fHbp elicits a robust bactericidal antibody response that correlates with the amount of fHbp expressed on the bacterial surface. fHbp is able to selectively bind human factor H, the key regulator of the alternative complement pathway, and this has important implications both for meningococcal pathogenesis and for vaccine design. Here, we review the functional and structural properties of fHbp, the strategies that led to the design of the two fHbp-based vaccines and the data generated during clinical studies.
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http://dx.doi.org/10.1586/14760584.2015.1016915DOI Listing
June 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

Genetically detoxified pertussis toxin (PT-9K/129G): implications for immunization and vaccines.

Expert Rev Vaccines 2014 Oct 3;13(10):1191-204. Epub 2014 Sep 3.

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

Pertussis toxin (PT) is one of the major virulence factors of Bordetella pertussis and the primary component of all pertussis vaccines available to date. Because of its various noxious effects the toxin needs to be detoxified. In all currently available vaccines, detoxification is achieved by treatment with high quantity of chemical agents such as formaldehyde, glutaraldehyde or hydrogen peroxide. Although effective in detoxification, this chemical treatment alters dramatically the immunological properties of the toxin. In contrast, PT genetically detoxified through the substitution of two residues necessary for its enzymatic activity maintains all functional and immunological properties. This review describes in detail the characteristics of this PT-9K/129G mutant and shows that it is non-toxic and a superior immunogen compared with chemically detoxified PT. Importantly, data from an efficacy trial show that the PT-9K/129G-based vaccine induces earlier and longer-lasting protection, further supporting the hypothesis that PT-9K/129G represents an ideal candidate for future pertussis vaccine formulations.
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http://dx.doi.org/10.1586/14760584.2014.942641DOI Listing
October 2014

Recombinant Clostridium difficile toxin fragments as carrier protein for PSII surface polysaccharide preserve their neutralizing activity.

Toxins (Basel) 2014 Apr 22;6(4):1385-96. Epub 2014 Apr 22.

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

Clostridium difficile is a Gram-positive bacterium and is the most commonly diagnosed cause of hospital-associated and antimicrobial-associated diarrhea. Despite the emergence of epidemic C. difficile strains having led to an increase in the incidence of the disease, a vaccine against this pathogen is not currently available. C. difficile strains produce two main toxins (TcdA and TcdB) and express three highly complex cell-surface polysaccharides (PSI, PSII and PSIII). PSII is the more abundantly expressed by most C. difficile ribotypes offering the opportunity of the development of a carbohydrate-based vaccine. In this paper, we evaluate the efficacy, in naive mice model, of PSII glycoconjugates where recombinant toxins A and B fragments (TcdA_B2 and TcdB_GT respectively) have been used as carriers. Both glycoconjugates elicited IgG titers anti-PSII although only the TcdB_GT conjugate induced a response comparable to that obtained with CRM197. Moreover, TcdA_B2 and TcdB_GT conjugated to PSII retained the ability to elicit IgG with neutralizing activity against the respective toxins. These results are a crucial proof of concept for the development of glycoconjugate vaccines against C. difficile infection (CDI) that combine different C. difficile antigens to potentially prevent bacterial colonization of the gut and neutralize toxin activity.
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http://dx.doi.org/10.3390/toxins6041385DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4014741PMC
April 2014

Vaccines against Clostridium difficile.

Hum Vaccin Immunother 2014 17;10(6):1466-77. Epub 2014 Mar 17.

Novartis Vaccines S.r.L.; Siena, Italy.

Clostridium difficile infection (CDI) is recognized as a major cause of nosocomial diseases ranging from antibiotic related diarrhea to fulminant colitis. Emergence during the last 2 decades of C. difficile strains associated with high incidence, severity and lethal outcomes has increased the challenges for CDI treatment. A limited number of drugs have proven to be effective against CDI and concerns about antibiotic resistance as well as recurring disease solicited the search for novel therapeutic strategies. Active vaccination provides the attractive opportunity to prevent CDI, and intense research in recent years led to development of experimental vaccines, 3 of which are currently under clinical evaluation. This review summarizes recent achievements and remaining challenges in the field of C. difficile vaccines, and discusses future perspectives in view of newly-identified candidate antigens.
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http://dx.doi.org/10.4161/hv.28428DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5396221PMC
June 2015

Vaccination against Clostridium difficile using toxin fragments: Observations and analysis in animal models.

Gut Microbes 2014 Mar-Apr;5(2):225-32. Epub 2014 Jan 22.

Institute of Infection, Immunity, and Inflammation; College of Medicine, Veterinary and Life Sciences; University of Glasgow; Glasgow, UK.

Clostridium difficile is a major cause of antibiotic associated diarrhea. Recently, we have shown that effective protection can be mediated in hamsters through the inclusion of specific recombinant fragments from toxin A and B in a systemically delivered vaccine. Interestingly while neutralizing antibodies to the binding domains of both toxin A and B are moderately protective, enhanced survival is observed when fragments from the glucosyltransferase region of toxin B replace those from the binding domain of this toxin. In this addendum, we discuss additional information that has been derived from such vaccination studies. This includes observations on efficacy and cross-protection against different ribotypes mediated by these vaccines and the challenges that remain for a vaccine which prevents clinical symptoms but not colonization. The use and value of vaccination both in the prevention of infection and for treatment of disease relapse will be discussed.
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http://dx.doi.org/10.4161/gmic.27712DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4063849PMC
February 2015

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

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

Structural vaccinology: a three-dimensional view for vaccine development.

Curr Top Med Chem 2013 ;13(20):2629-37

Novartis Vaccines and Diagnostics, Siena, Italy.

The Structural Vaccinology (SV) approach is the logical evolution of Reverse Vaccinology: a genome-based approach combined with structural biology, with the idea that protective determinants can be used to selectively engineer the antigens that can be re-designed and simplified for inclusion in vaccine combinations. The final objectives of the rational structure-based antigen optimization are the facilitation of industrial-scale production of the antigens combination, obtain a greater immunogenicity and a greater safety profile and finally expand the breadth of protection. Structural Vaccinology is particularly powerful in case of antigenic variation between closely related strains and species. Several examples are available in literature of how SV has already been applied successfully to several bacterial and viral projects. The examples of structure-based antigens optimization reviewed here describe different template procedures that can be followed to develop improved vaccines against other pathogens and potentially help resolve challenges in manufacturing or efficacy.
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http://dx.doi.org/10.2174/15680266113136660187DOI Listing
June 2014

Diversity of cwp loci in clinical isolates of Clostridium difficile.

J Med Microbiol 2013 Sep 30;62(Pt 9):1444-1452. Epub 2013 May 30.

Research Centre, NovartisV&D, Siena, Italy.

An increased incidence of Clostridium difficile infection (CDI) is associated with the emergence of epidemic strains characterized by high genetic diversity. Among the factors that may have a role in CDI is a family of 29 paralogues, the cell-wall proteins (CWPs), which compose the outer layer of the bacterial cell and are likely to be involved in colonization. Previous studies have shown that 12 of the 29 cwp genes are clustered in the same region, named after slpA (cwp1), the slpA locus, whereas the remaining 17 paralogues are distributed throughout the genome. The variability of 14 of these 17 cwp paralogues was determined in 40 C. difficile clinical isolates belonging to six of the currently prevailing PCR ribotypes. Based on sequence conservation, these cwp genes were divided into two groups, one comprising nine cwp loci having highly conserved sequences in all isolates, and the other five loci showing low genetic conservation among isolates of the same PCR ribotype, as well as between different PCR ribotypes. Three conserved CWPs, Cwp16, Cwp18 and Cwp25, and two variable ones, Cwp26 and Cwp27, were characterized further by Western blot analysis of total cell extracts or surface-layer preparations of the C. difficile clinical isolates. Expression of genetically invariable CWPs was well conserved in all isolates, whilst genetically variable CWPs were not always expressed at comparable levels, even in strains containing identical sequences but belonging to different PCR ribotypes. This is the first report on the distribution and variability of a number of genes encoding CWPs in C. difficile.
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http://dx.doi.org/10.1099/jmm.0.058719-0DOI Listing
September 2013

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

CbpA: a novel surface exposed adhesin of Clostridium difficile targeting human collagen.

Cell Microbiol 2013 Oct 11;15(10):1674-87. Epub 2013 Apr 11.

Novartis Vaccines and Diagnostics Srl, Siena, Italy.

Clostridium difficile is the leading cause of antibiotic-associated diarrhoea and pseudomembranous colitis. While the role of toxins in pathogenesis has been extensively described, the contribution of surface determinants to intestinal colonization is still poorly understood. We focused our study on a novel member of the MSCRAMM family, named CbpA (Collagen binding protein A), for its adhesive properties towards collagen. We demonstrate that CbpA, which carries an LPXTG-like cell wall anchoring domain, is expressed on the bacterial surface of C. difficile and that the recombinant protein binds at high affinity to collagens I and V (apparent Kd in the order of 10(-9 ) M). These findings were validated by confocal microscopy studies showing the colocalization of the protein with type I and V collagen fibres produced by human fibroblasts and mouse intestinal tissues. However, the collagen binding activity of the wild-type C. difficile 630 strain was indistinguishable to the cbpA knock-out strain. To overcome this apparent clostridial adherence redundancy, we engineered a Lactococcus lactis strain for the heterologous expression of CbpA. When exposed on the surface of L. lactis, CbpA significantly enhances the ability of the bacterium to interact with collagen and to adhere to ECM-producing cells. The binding activity of L. lactis-CbpA strain was prevented by an antiserum raised against CbpA, demonstrating the specificity of the interaction. These results suggest that CbpA is a newsurface-exposed adhesin contributing to the C. difficile interaction with the host.
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http://dx.doi.org/10.1111/cmi.12139DOI Listing
October 2013

Targeted amino acid substitutions impair streptolysin O toxicity and group A Streptococcus virulence.

mBio 2013 Jan 8;4(1):e00387-12. Epub 2013 Jan 8.

Novartis Vaccines and Diagnostics, Siena, Italy.

Unlabelled: Streptolysin O is a potent pore-forming toxin produced by group A Streptococcus. The aims of the present study were to dissect the relative contributions of different structural domains of the protein to hemolytic activity, to obtain a detoxified form of streptolysin O amenable to human vaccine formulation, and to investigate the role of streptolysin O-specific antibodies in protection against group A Streptococcus infection. On the basis of in silico structural predictions, we introduced two amino acid substitutions, one in the proline-rich domain 1 and the other in the conserved undecapeptide loop in domain 4. The resulting streptolysin O derivative showed no toxicity, was highly impaired in binding to eukaryotic cells, and was unable to form organized oligomeric structures on the cell surface. However, it was fully capable of conferring consistent protection in a murine model of group A Streptococcus infection. When we engineered a streptococcal strain to express the double-mutated streptolysin O, a drastic reduction in virulence as well as a diminished capacity to kill immune cells recruited at the infection site was observed. Furthermore, when mice immunized with the toxoid were challenged with the wild-type and mutant strains, protection only against the wild-type strain, not against the strain expressing the double-mutated streptolysin O, was obtained. We conclude that protection occurs by antibody-mediated neutralization of active toxin.

Importance: We present a novel example of structural design of a vaccine antigen optimized for human vaccine use. Having previously demonstrated that immunization of mice with streptolysin O elicits a protective immune response against infection with group A Streptococcus strains of different serotypes, we developed in this study a double-mutated nontoxic derivative that represents a novel tool for the development of protective vaccine formulations against this important human pathogen. Furthermore, the innovative construction of an isogenic strain expressing a functionally inactive toxin and its use in infection and opsonophagocytosis experiments allowed us to investigate the mechanism by which streptolysin O mediates protection against group A Streptococcus. Finally, the ability of this toxin to directly attack and kill host immune cells during infection was studied in an air pouch model, which allowed parallel quantification of cellular recruitment, vitality, and cytokine release at the infection site.
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http://dx.doi.org/10.1128/mBio.00387-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3546560PMC
January 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

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 insight into the mechanism of DNA-binding attenuation of the Neisserial adhesin repressor NadR by the small natural ligand 4-hydroxyphenylacetic acid.

Biochemistry 2012 Aug 15;51(34):6738-52. Epub 2012 Aug 15.

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

Neisserial adhesin A (NadA) is a surface exposed trimeric protein present in most hypervirulent meningococcal strains and involved in epithelial cell adhesion and colonization. The expression of nadA is controlled by Neisserial adhesin regulator (NadR), a member of the MarR family, which binds to the nadA promoter and strongly represses the transcription of nadA. It was recently demonstrated that the DNA-binding activity of NadR was attenuated by 4-hydroxyphenylacetic acid (4-HPA), a natural molecule released in human saliva, thus leading to the de-repression of nadA in vivo. To elucidate the mechanism of regulation of NadR by 4-HPA, we used hydrogen-deuterium exchange mass spectrometry in association with in silico docking and site-directed mutagenesis. We show here that 4-HPA binds at the interface between the dimerization and the DNA-binding domains and stabilizes the homodimeric state of NadR without inducing large conformational changes in the DNA-binding lobes. The residues predicted to be in contact with 4-HPA were further selected for mutagenesis to assess their in vitro and in vivo functions in 4-HPA binding. Our results indicate that Arg(40) is critical for DNA-binding and reveal that Tyr(115) plays a key role in the mechanism of regulation of NadR by 4-HPA. Altogether our data suggest that the mechanism of regulation of NadR by 4-HPA mainly involves the stabilization of the dimer in a configuration incompatible with DNA binding.
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http://dx.doi.org/10.1021/bi300656wDOI Listing
August 2012