Publications by authors named "Daniele Veggi"

34 Publications

Rational design of adjuvants for subunit vaccines: The format of cationic adjuvants affects the induction of antigen-specific antibody responses.

J Control Release 2021 Feb 2;330:933-944. Epub 2020 Nov 2.

Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow G4 0RE, UK. Electronic address:

A range of cationic delivery systems have been investigated as vaccine adjuvants, though few direct comparisons exist. To investigate the impact of the delivery platform, we prepared four cationic systems (emulsions, liposomes, polymeric nanoparticles and solid lipid nanoparticles) all containing equal concentrations of the cationic lipid dimethyldioctadecylammonium bromide in combination with the Neisseria adhesin A variant 3 subunit antigen. The formulations were physicochemically characterized and their ability to associate with cells and promote antigen processing (based on degradation of DQ-OVA, a substrate for proteases which upon hydrolysis is fluorescent) was compared in vitro and their vaccine efficacy (antigen-specific antibody responses and IFN-γ production) and biodistribution (antigen and adjuvant) were evaluated in vivo. Due to their cationic nature, all delivery systems gave high antigen loading (> 85%) with liposomes, lipid nanoparticles and emulsions being <200 nm, whilst polymeric nanoparticles were larger (~350 nm). In vitro, the particulate systems tended to promote cell uptake and antigen processing, whilst emulsions were less effective. Similarly, whilst the particulate delivery systems induced a depot (of both delivery system and antigen) at the injection site, the cationic emulsions did not. However, out of the systems tested the cationic emulsions induced the highest antibody responses. These results demonstrate that while cationic lipids can have strong adjuvant activity, their formulation platform influences their immunogenicity.
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http://dx.doi.org/10.1016/j.jconrel.2020.10.066DOI Listing
February 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

Cocrystal structure of meningococcal factor H binding protein variant 3 reveals a new crossprotective epitope recognized by human mAb 1E6.

FASEB J 2019 11 5;33(11):12099-12111. Epub 2019 Oct 5.

GlaxoSmithKline, Siena, Italy.

The 4 component meningococcus B vaccine (4CMenB) vaccine is the first vaccine containing recombinant proteins licensed for the prevention of invasive meningococcal disease caused by meningococcal serogroup B strains. 4CMenB contains 3 main recombinant proteins, including the factor H binding protein (fHbp), a lipoprotein able to bind the human factor H. To date, over 1000 aa sequences of fHbp have been identified, and they can be divided into variant groups 1, 2, and 3, which are usually not crossprotective. Nevertheless, previous characterizations of a small set ( = 10) of mAbs generated in humans after 4CMenB immunization revealed 2 human Fabs (huFabs) (1A12, 1G3) with some crossreactivity for variants 1, 2, and 3. This unexpected result prompted us to examine a much larger set of human mAbs ( = 110), with the aim of better understanding the extent and nature of crossreactive anti-fHbp antibodies. In this study, we report an analysis of the human antibody response to fHbp, by the characterization of 110 huFabs collected from 3 adult vaccinees during a 6-mo study. Although the 4CMenB vaccine contains fHbp variant 1, 13 huFabs were also found to be crossreactive with variants 2 and 3. The crystal structure of the crossreactive huFab 1E6 in complex with fHbp variant 3 was determined, revealing a novel, highly conserved epitope distinct from the epitopes recognized by 1A12 or 1G3. Further, functional characterization shows that human mAb 1E6 is able to elicit rabbit, but not human, complement-mediated bactericidal activity against meningococci displaying fHbp from any of the 3 different variant groups. This functional and structural information about the human antibody response upon 4CMenB immunization contributes to further unraveling the immunogenic properties of fHbp. Knowledge gained about the epitope profile recognized by the human antibody repertoire could guide future vaccine design.-Bianchi, F., Veggi, D., Santini, L., Buricchi, F., Bartolini, E., Lo Surdo, P., Martinelli, M., Finco, O., Masignani, V., Bottomley, M. J., Maione, D., Cozzi, R. Cocrystal structure of meningococcal factor H binding protein variant 3 reveals a new crossprotective epitope recognized by human mAb 1E6.
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http://dx.doi.org/10.1096/fj.201900374RDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6902690PMC
November 2019

Structures of NHBA elucidate a broadly conserved epitope identified by a vaccine induced antibody.

PLoS One 2018 22;13(8):e0201922. Epub 2018 Aug 22.

GSK, Rockville, MD, United States of America.

Neisserial heparin binding antigen (NHBA) is one of three main recombinant protein antigens in 4CMenB, a vaccine for the prevention of invasive meningococcal disease caused by Neisseria meningitidis serogroup B. NHBA is a surface-exposed lipoprotein composed of a predicted disordered N-terminal region, an arginine-rich region that binds heparin, and a C-terminal domain that folds as an anti-parallel β-barrel and that upon release after cleavage by human proteases alters endothelial permeability. NHBA induces bactericidal antibodies in humans, and NHBA-specific antibodies elicited by the 4CMenB vaccine contribute to serum bactericidal activity, the correlate of protection. To better understand the structural bases of the human antibody response to 4CMenB vaccination and to inform antigen design, we used X-ray crystallography to elucidate the structures of two C-terminal fragments of NHBA, either alone or in complex with the Fab derived from the vaccine-elicited human monoclonal antibody 5H2, and the structure of the unbound Fab 5H2. The structures reveal details on the interaction between an N-terminal β-hairpin fragment and the β-barrel, and explain how NHBA is capable of generating cross-reactive antibodies through an extensive conserved conformational epitope that covers the entire C-terminal face of the β-barrel. By providing new structural information on a vaccine antigen and on the human immune response to vaccination, these results deepen our molecular understanding of 4CMenB, and might also aid future vaccine design projects.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0201922PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6104945PMC
January 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

Crystal structures of human Fabs targeting the Bexsero meningococcal vaccine antigen NHBA.

Acta Crystallogr F Struct Biol Commun 2017 06 11;73(Pt 6):305-314. Epub 2017 May 11.

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

Neisserial heparin-binding antigen (NHBA) is a surface-exposed lipoprotein from Neisseria meningitidis and is a component of the meningococcus B vaccine Bexsero. As part of a study to characterize the three-dimensional structure of NHBA and the molecular basis of the human immune response to Bexsero, the crystal structures of two fragment antigen-binding domains (Fabs) isolated from human monoclonal antibodies targeting NHBA were determined. Through a high-resolution analysis of the organization and the amino-acid composition of the CDRs, these structures provide broad insights into the NHBA epitopes recognized by the human immune system. As expected, these Fabs also show remarkable structural conservation, as shown by a structural comparison of 15 structures of apo Fab 10C3 which were obtained from crystals grown in different crystallization conditions and were solved while searching for a complex with a bound NHBA fragment or epitope peptide. This study also provides indirect evidence for the intrinsically disordered nature of two N-terminal regions of NHBA.
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http://dx.doi.org/10.1107/S2053230X17006021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5458386PMC
June 2017

Structure of a protective epitope of group B type III capsular polysaccharide.

Proc Natl Acad Sci U S A 2017 05 24;114(19):5017-5022. Epub 2017 Apr 24.

GSK Vaccines, 53100 Siena, Italy

Despite substantial progress in the prevention of group B (GBS) disease with the introduction of intrapartum antibiotic prophylaxis, this pathogen remains a leading cause of neonatal infection. Capsular polysaccharide conjugate vaccines have been tested in phase I/II clinical studies, showing promise for further development. Mapping of epitopes recognized by protective antibodies is crucial for understanding the mechanism of action of vaccines and for enabling antigen design. In this study, we report the structure of the epitope recognized by a monoclonal antibody with opsonophagocytic activity and representative of the protective response against type III GBS polysaccharide. The structure and the atomic-level interactions were determined by saturation transfer difference (STD)-NMR and X-ray crystallography using oligosaccharides obtained by synthetic and depolymerization procedures. The GBS PSIII epitope is made by six sugars. Four of them derive from two adjacent repeating units of the PSIII backbone and two of them from the branched galactose-sialic acid disaccharide contained in this sequence. The sialic acid residue establishes direct binding interactions with the functional antibody. The crystal structure provides insight into the molecular basis of antibody-carbohydrate interactions and confirms that the conformational epitope is not required for antigen recognition. Understanding the structural basis of immune recognition of capsular polysaccharide epitopes can aid in the design of novel glycoconjugate vaccines.
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http://dx.doi.org/10.1073/pnas.1701885114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5441712PMC
May 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

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

Neisseria meningitis GNA1030 is a ubiquinone-8 binding protein.

FASEB J 2015 Jun 20;29(6):2260-7. Epub 2015 Feb 20.

Novartis Vaccines, Siena, Italy

Bexsero, a new vaccine against Neisseria meningitidis serogroup B (MenB), is composed of 3 main recombinant proteins and an outer membrane vesicle component. One of the main bactericidal antigens, neisseria heparin binding antigen (NHBA), is present as a fusion protein with the accessory protein genome-derived neisserial antigen (GNA) 1030 to further increase its immunogenicity. The gene encoding for GNA1030 is present and highly conserved in all Neisseria strains, and although orthologs are present in numerous species, its biologic function is unknown. Native mass spectrometry was used to demonstrate that GNA1030 forms a homodimer associated with 2 molecules of ubiquinone-8 (Ub8), a cofactor mainly involved in the electron transport chain and with antioxidant properties. Disc diffusion assays on the wild-type and knockout mutant of GNA1030, in the presence of various compounds, suggested that GNA1030 is not involved in oxidative stress or electron chain transport per se, although it contributes to constitutive refilling of the inner membrane with Ub8. These studies shed light on an accessory protein present in Bexsero and reveal functional insights into the family of related proteins. On the basis of our findings, we propose to name the protein neisseria ubiquinone binding protein (NUbp).
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http://dx.doi.org/10.1096/fj.14-263954DOI 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

Auto ADP-ribosylation of NarE, a Neisseria meningitidis ADP-ribosyltransferase, regulates its catalytic activities.

FASEB J 2013 Dec 20;27(12):4723-30. Epub 2013 Aug 20.

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

NarE is an arginine-specific mono-ADP-ribosyltransferase identified in Neisseria meningitidis that requires the presence of iron in a structured cluster for its enzymatic activities. In this study, we show that NarE can perform auto-ADP-ribosylation. This automodification occurred in a time- and NAD-concentration-dependent manner; was inhibited by novobiocin, an ADP-ribosyltransferase inhibitor; and did not occur when NarE was heat inactivated. No reduction in incorporation was evidenced in the presence of high concentrations of ATP, GTP, ADP-ribose, or nicotinamide, which inhibits NAD-glycohydrolase, impeding the formation of free ADP-ribose. Based on the electrophoretic profile of NarE on auto-ADP-ribosylation and on the results of mutagenesis and mass spectrometry analysis, the auto-ADP-ribosylation appeared to be restricted to the addition of a single ADP-ribose. Chemical stability experiments showed that the ADP-ribosyl linkage was sensitive to hydroxylamine, which breaks ADP-ribose-arginine bonds. Site-directed mutagenesis suggested that the auto-ADP-ribosylation site occurred preferentially on the R(7) residue, which is located in the region I of the ADP-ribosyltransferase family. After auto-ADP-ribosylation, NarE showed a reduction in ADP-ribosyltransferase activity, while NAD-glycohydrolase activity was increased. Overall, our findings provide evidence for a novel intramolecular mechanism used by NarE to regulate its enzymatic activities.
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http://dx.doi.org/10.1096/fj.13-229955DOI Listing
December 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

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

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

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

FdeC, a novel broadly conserved Escherichia coli adhesin eliciting protection against urinary tract infections.

mBio 2012 10;3(2). Epub 2012 Apr 10.

Novartis Vaccines and Diagnostics Srl, Siena, Italy.

Unlabelled: The increasing antibiotic resistance of pathogenic Escherichia coli species and the absence of a pan-protective vaccine pose major health concerns. We recently identified, by subtractive reverse vaccinology, nine Escherichia coli antigens that protect mice from sepsis. In this study, we characterized one of them, ECOK1_0290, named FdeC (factor adherence E. coli) for its ability to mediate E. coli adhesion to mammalian cells and extracellular matrix. This adhesive propensity was consistent with the X-ray structure of one of the FdeC domains that shows a striking structural homology to Yersinia pseudotuberculosis invasin and enteropathogenic E. coli intimin. Confocal imaging analysis revealed that expression of FdeC on the bacterial surface is triggered by interaction of E. coli with host cells. This phenotype was also observed in bladder tissue sections derived from mice infected with an extraintestinal strain. Indeed, we observed that FdeC contributes to colonization of the bladder and kidney, with the wild-type strain outcompeting the fdeC mutant in cochallenge experiments. Finally, intranasal mucosal immunization with recombinant FdeC significantly reduced kidney colonization in mice challenged transurethrally with uropathogenic E. coli, supporting a role for FdeC in urinary tract infections.

Importance: Pathogenic Escherichia coli strains are involved in a diverse spectrum of diseases, including intestinal and extraintestinal infections (urinary tract infections and sepsis). The absence of a broadly protective vaccine against all these E. coli strains is a major problem for modern society due to high costs to health care systems. Here, we describe the structural and functional properties of a recently reported protective antigen, named FdeC, and elucidated its putative role during extraintestinal pathogenic E. coli infection by using both in vitro and in vivo infection models. The conservation of FdeC among strains of different E. coli pathotypes highlights its potential as a component of a broadly protective vaccine against extraintestinal and intestinal E. coli infections.
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http://dx.doi.org/10.1128/mBio.00010-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3324786PMC
June 2012

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

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

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

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

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

Structure of the uncomplexed Neisseria meningitidis factor H-binding protein fHbp (rLP2086).

Acta Crystallogr Sect F Struct Biol Cryst Commun 2011 May 20;67(Pt 5):531-5. Epub 2011 Apr 20.

Department of Biological Chemistry, University of Padua, Viale G. Colombo 3, 35121 Padua, Italy.

fHbp, a highly immunogenic outer membrane protein of Neisseria meningitidis, is responsible for binding to human factor H, a multi-domain protein which is the central regulator of the alternative complement pathway. Here, the crystal structure of mature fHbp determined at 2 Å resolution is presented and is compared with the structure of the same protein in complex with factor H domains 6 and 7 recently solved using X-ray techniques. While the overall protein fold is well conserved, modifications are observed mainly in the loop regions involved in the interaction, reflecting a specific adaptation of fHbp in complexing factor H with high affinity. Such a comparison has to date been impaired by the fact that fHbp models determined by NMR show remarkable differences over the entire structure.
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http://dx.doi.org/10.1107/S1744309111006154DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3087634PMC
May 2011

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

Structural and biochemical characterization of NarE, an iron-containing ADP-ribosyltransferase from Neisseria meningitidis.

J Biol Chem 2011 Apr 2;286(17):14842-51. Epub 2011 Mar 2.

Bijvoet Center for Biomolecular Research, Science Faculty, Utrecht University, Padualaan 8, 3584 CH, Utrecht, The Netherlands.

NarE is a 16 kDa protein identified from Neisseria meningitidis, one of the bacterial pathogens responsible for meningitis. NarE belongs to the family of ADP-ribosyltransferases (ADPRT) and catalyzes the transfer of ADP-ribose moieties to arginine residues in target protein acceptors. Many pathogenic bacteria utilize ADP-ribosylating toxins to modify and alter essential functions of eukaryotic cells. NarE is further the first ADPRT which could be shown to bind iron through a Fe-S center, which is crucial for the catalytic activity. Here we present the NMR solution structure of NarE, which shows structural homology to other ADPRTs. Using NMR titration experiments we could identify from Chemical Shift Perturbation data both the NAD binding site, which is in perfect agreement with a consensus sequence analysis between different ADPRTs, as well as the iron coordination site, which consists of 2 cysteines and 2 histidines. This atypical iron coordination is also capable to bind zinc. These results could be fortified by site-directed mutagenesis of the catalytic region, which identified two functionally crucial residues. We could further identify a main interaction region of NarE with antibodies using two complementary methods based on antibody immobilization, proteolytic digestion, and mass spectrometry. This study combines structural and functional features of NarE providing for the first time a characterization of an iron-dependent ADPRT.
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http://dx.doi.org/10.1074/jbc.M110.193623DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3083161PMC
April 2011

NMR resonance assignments of NarE, a putative ADP-ribosylating toxin from Neisseria meningitidis.

Biomol NMR Assign 2011 Apr 25;5(1):35-8. Epub 2010 Aug 25.

Bijvoet Center for Biomolecular Research, Science Faculty, Utrecht University, The Netherlands.

NarE is a 16 kDa protein identified from Neisseria meningitidis, one of the bacterial pathogens responsible for meningitis. NarE belongs to the ADP-ribosyltransferase family and catalyses the transfer of ADP-ribose moieties to arginine residues in target protein acceptors. Many pathogenic bacteria utilize ADP-ribosylating toxins to modify and alter essential functions of eukaryotic cells. NarE was proposed to bind iron through a Fe-S center which is supposed to be implied in catalysis. We have produced and purified uniformly labeled (15)N- and (15)N/(13)C-NarE and assigned backbone and side-chain resonances using multidimensional heteronuclear NMR spectroscopy. These assignments provide the starting point for the three-dimensional structure determination of NarE and the characterization of the role of the Fe-S center in the catalytic mechanism.
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http://dx.doi.org/10.1007/s12104-010-9261-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3049222PMC
April 2011

1H, 13C and 15N assignment of the C-terminal domain of GNA2132 from Neisseria meningitidis.

Biomol NMR Assign 2010 Apr 20;4(1):107-9. Epub 2010 Mar 20.

National Institute for Medical Research, The Ridgeway, London, UK.

GNA2132 (Genome-derived Neisseria Antigen 2132) is a surface-exposed lipoprotein discovered by reverse vaccinology and expressed by genetically diverse Neisseria meningitidis strains (Pizza et al. 2000). The protein induces bactericidal antibodies against most strains of Meningococccus and has been included in a multivalent recombinant vaccine against N. meningitidis serogroup B. Structure determination of GNA2132 is important for understanding the antigenic properties of the protein in view of increased efficiency vaccine development. We report practically complete (1)H, (13)C and (15)N assignment of the detectable spectrum of a highly conserved C-terminal region of GNA2132 (residues 245-427) in micellar solution, a medium used to improve the spectral quality. The first 32 residues of our construct up to residue 277 were not visible in the spectrum, presumably because of line broadening due to solvent and/or conformational exchange. Secondary structure predictions based on chemical shift information indicate the presence of an all beta-protein with eight beta strands.
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http://dx.doi.org/10.1007/s12104-010-9220-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2862177PMC
April 2010

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

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

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

Neisseria meningitidis NhhA is a multifunctional trimeric autotransporter adhesin.

Mol Microbiol 2006 Aug 27;61(3):631-44. Epub 2006 Jun 27.

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

NhhA, Neisseriahia/hsf homologue, or GNA0992, is an oligomeric outer membrane protein of Neisseria meningitidis, recently included in the family of trimeric autotransporter adhesins. In this study we present the structural and functional characterization of this protein. By expressing in Escherichia coli the full-length gene, deletion mutants and chimeric proteins of NhhA, we demonstrated that the last 72 C-terminal residues are able to allow trimerization and localization of the N-terminal protein domain to the bacterial surface. In addition, we investigated on the possible role of NhhA in bacterial-host interaction events. We assessed in vitro the ability of recombinant purified NhhA to bind human epithelial cells as well as laminin and heparan sulphate. Furthermore, we shown that E. coli strain expressing NhhA was able to adhere to epithelial cells, and observed a reduced adherence in a meningococcal isogenic MC58DeltaNhhA mutant. We concluded that this protein is a multifunctional adhesin, able to promote the bacterial adhesion to host cells and extracellular matrix components. Collectively, our results underline a putative role of NhhA in meningococcal pathogenesis and ascertain its structural and functional belonging to the emerging group of bacterial autotransporter adhesins with trimeric architecture.
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http://dx.doi.org/10.1111/j.1365-2958.2006.05261.xDOI Listing
August 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