Publications by authors named "Vega Masignani"

65 Publications

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

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

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

The Development of a Vaccine Against Meningococcus B Using Reverse Vaccinology.

Front Immunol 2019 16;10:751. Epub 2019 Apr 16.

Department of Pediatrics, Oxford University, Oxford, United Kingdom.

The discovery of vaccine antigens through whole genome sequencing (WGS) contrasts with the classical hypothesis-driven laboratory-based analysis of microbes to identify components to elicit protective immunity. This radical change in scientific direction and action in vaccine research is captured in the term . The complete genome sequence of an isolate of serogroup B (MenB) was systematically analyzed to identify proteins predicted to be secreted or exported to the outer membrane. This identified hundreds of genes coding for potential surface-exposed antigens. These were amplified, cloned in expression vectors and used to immunize mice. Antisera against 350 recombinant antigens were obtained and analyzed in a panel of immunological assays from which 28 were selected as potentially protective based on the -antibody dependent, complement mediated- serum bactericidal activity assay. Testing of these candidate vaccine antigens, using a large globally representative strain collection of Neisseria species isolated from cases of disease and carriage, indicated that no single component would be sufficient to induce broad coverage and that a "universal" vaccine should contain multiple antigens. The final choice of antigens to be included was based on cross-protective ability, assayed by serum bactericidal activity and maximum coverage of the extensive antigenic variability of MenB strains. The resulting multivalent vaccine formulation selected consisted of three recombinant antigens (Neisserial Heparin Binding Antigen or NHBA, Factor H binding protein or fHbp and Neisseria Adhesin A or NadA). To improve immunogenicity and potential strain coverage, an outer membrane vesicle component obtained from the epidemic New Zealand strain (OMVNz) was added to the formulation to create a four component vaccine, called 4CMenB. A series of phase 2 and 3 clinical trials were conducted to evaluate safety and tolerability and to estimate the vaccine effectiveness of human immune responses at different ages and how these were affected by various factors including concomitant vaccine use and lot-to-lot consistency. 4CMenB was approved in Europe in 2013 and introduced in the National Immunization Program in the UK starting from September 2015 when the vaccine was offered to all newborns using a 2, 4, and 12 months schedule., The effectiveness against invasive MenB disease measured at 11 months after the study start and 5 months after the second vaccination was 83% and there have been no safety concerns.
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http://dx.doi.org/10.3389/fimmu.2019.00751DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6477034PMC
September 2020

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

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

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

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

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

Meningococcal B vaccine (4CMenB): the journey from research to real world experience.

Expert Rev Vaccines 2018 12 5;17(12):1111-1121. Epub 2018 Dec 5.

d Vaccine Development Leader , Research and Development Centre , Rockville , MD , USA.

Introduction: Neisseria meningitidis serogroup B (MenB) is the most common cause of bacterial meningitis in many industrialized countries and occurs at any age. The highest incidence is in infants aged <1 year, followed by children and adolescents. Four-component MenB vaccine (4CMenB, Bexsero) is the only MenB vaccine authorized for use in all age-groups. Experience with 4CMenB is growing as it is implemented in different countries/age-groups encompassing university students, children, adolescents, and infant mass vaccination programs.

Areas Covered: An update of recently available data describing the mechanism of immunogenicity of 4CMenB and real-world evidence of vaccine effectiveness and disease impact. We discuss the appropriate age for vaccination to maximize population impacts.

Expert Commentary: Invasive meningococcal disease is uncommon and sufficiently powered efficacy studies were not feasible during 4CMenB development. Additionally, several thousand genetically diverse invasive MenB strains circulate globally, varying widely in surface protein expression. This posed significant challenges in predicting clinical protection with MenB vaccines. Five years of 4CMenB use post-licensure confirm the clinical benefit of vaccination as predicted during development. Preliminary evidence suggests an extended impact on other meningococcal serogroups and Neisseria gonorrhoeae.
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http://dx.doi.org/10.1080/14760584.2018.1547637DOI Listing
December 2018

Lectin activity of the pneumococcal pilin proteins.

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

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

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

Emerging experience with meningococcal serogroup B protein vaccines.

Expert Rev Vaccines 2017 May 10;16(5):433-451. Epub 2017 Apr 10.

d Research & Development, GSK , Siena , Italy.

Introduction: The successful development of two broadly protective vaccines targeting Neisseria meningitidis serogroup B (MenB); 4CMenB and rLP2086, is the most significant recent advance in meningococcal disease prevention. Areas covered: Here we review the principles underlying the development of each vaccine and the novel methods used to estimate vaccine coverage. We update clinical and post-licensure experience with 4CMenB and rLP2086. Expert commentary: The immunogenicity and acceptable safety profile of 4CMenB and rLP2086 has been demonstrated in clinical trials. Continuing uncertainties exist around the appropriate age groups to be immunized, the degree and duration of efficacy, and the impact on nasopharyngeal carriage which has implications for strategies to interrupt transmission and maximize herd protection effects. Universal vaccination programs such as those undertaken in Quebec and the United Kingdom are providing important information on these issues. The potential for MenB vaccines to prevent infection by other serogroups appears promising, and the impact of MenB vaccines on other pathogenic neisserial species with similar surface proteins warrants further investigation.
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http://dx.doi.org/10.1080/14760584.2017.1308828DOI Listing
May 2017

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

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

GSK Vaccines, Siena, Italy.

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

Functional characterization of a monoclonal antibody epitope using a lambda phage display-deep sequencing platform.

Sci Rep 2016 08 17;6:31458. Epub 2016 Aug 17.

Scylla Biotech Srl, Messina, Italy.

We have recently described a method, named PROFILER, for the identification of antigenic regions preferentially targeted by polyclonal antibody responses after vaccination. To test the ability of the technique to provide insights into the functional properties of monoclonal antibody (mAb) epitopes, we used here a well-characterized epitope of meningococcal factor H binding protein (fHbp), which is recognized by mAb 12C1. An fHbp library, engineered on a lambda phage vector enabling surface expression of polypeptides of widely different length, was subjected to massive parallel sequencing of the phage inserts after affinity selection with the 12C1 mAb. We detected dozens of unique antibody-selected sequences, the most enriched of which (designated as FrC) could largely recapitulate the ability of fHbp to bind mAb 12C1. Computational analysis of the cumulative enrichment of single amino acids in the antibody-selected fragments identified two overrepresented stretches of residues (H248-K254 and S140-G154), whose presence was subsequently found to be required for binding of FrC to mAb 12C1. Collectively, these results suggest that the PROFILER technology can rapidly and reliably identify, in the context of complex conformational epitopes, discrete "hot spots" with a crucial role in antigen-antibody interactions, thereby providing useful clues for the functional characterization of the epitope.
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http://dx.doi.org/10.1038/srep31458DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4987625PMC
August 2016

Epitope Mapping of a Monoclonal Antibody Directed against Neisserial Heparin Binding Antigen Using Next Generation Sequencing of Antigen-Specific Libraries.

PLoS One 2016 10;11(8):e0160702. Epub 2016 Aug 10.

Scylla Biotech Srl, Messina, Italy.

We explore here the potential of a newly described technology, which is named PROFILER and is based on next generation sequencing of gene-specific lambda phage-displayed libraries, to rapidly and accurately map monoclonal antibody (mAb) epitopes. For this purpose, we used a novel mAb (designated 31E10/E7) directed against Neisserial Heparin-Binding Antigen (NHBA), a component of the anti-group B meningococcus Bexsero® vaccine. An NHBA phage-displayed library was affinity-selected with mAb 31E10/E7, followed by massive sequencing of the inserts present in antibody-selected phage pools. Insert analysis identified an amino acid stretch (D91-A128) in the N-terminal domain, which was shared by all of the mAb-enriched fragments. Moreover, a recombinant fragment encompassing this sequence could recapitulate the immunoreactivity of the entire NHBA molecule against mAb 31E10/E7. These results were confirmed using a panel of overlapping recombinant fragments derived from the NHBA vaccine variant and a set of chemically synthetized peptides covering the 10 most frequent antigenic variants. Furthermore, hydrogen-deuterium exchange mass-spectrometry analysis of the NHBA-mAb 31E10/E7 complex was also compatible with mapping of the epitope to the D91-A128 region. Collectively, these results indicate that the PROFILER technology can reliably identify epitope-containing antigenic fragments and requires considerably less work, time and reagents than other epitope mapping methods.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0160702PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4980009PMC
August 2017

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

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

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

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

Phage display revisited: Epitope mapping of a monoclonal antibody directed against Neisseria meningitidis adhesin A using the PROFILER technology.

MAbs 2016 May-Jun;8(4):741-50. Epub 2016 Mar 10.

a Scylla Biotech Srl , Messina , Italy.

There is a strong need for rapid and reliable epitope mapping methods that can keep pace with the isolation of increasingly larger numbers of mAbs. We describe here the identification of a conformational epitope using Phage-based Representation OF ImmunoLigand Epitope Repertoire (PROFILER), a recently developed high-throughput method based on deep sequencing of antigen-specific lambda phage-displayed libraries. A novel bactericidal monoclonal antibody (mAb 9F11) raised against Neisseria meningitidis adhesin A (NadA), an important component of the Bexsero(®) anti-meningococcal vaccine, was used to evaluate the technique in comparison with other epitope mapping methods. The PROFILER technology readily identified NadA fragments that were capable of fully recapitulating the reactivity of the entire antigen against mAb 9F11. Further analysis of these fragments using mutagenesis and hydrogen-deuterium exchange mass-spectrometry allowed us to identify the binding site of mAb 9F11 (A250-D274) and an adjoining sequence (V275-H312) that was also required for the full functional reconstitution of the epitope. These data suggest that, by virtue of its ability to detect a great variety of immunoreactive antigen fragments in phage-displayed libraries, the PROFILER technology can rapidly and reliably identify epitope-containing regions and provide, in addition, useful clues for the functional characterization of conformational mAb epitopes.
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http://dx.doi.org/10.1080/19420862.2016.1158371DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4966859PMC
November 2017

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

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

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

GSK Vaccines, Siena.

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

Exploiting chimeric human antibodies to characterize a protective epitope of Neisseria adhesin A, one of the Bexsero vaccine components.

FASEB J 2016 Jan 24;30(1):93-101. Epub 2015 Aug 24.

Novartis Vaccines and Diagnostics, GlaxoSmithKline, Siena, Italy

Neisseria adhesin A (NadA) is one of the antigens of Bexsero, the recently licensed multicomponent vaccine against serogroup B Neisseria meningitidis (MenB). NadA belongs to the class of oligomeric coiled-coil adhesins and is able to mediate adhesion and invasion of human epithelial cells. As a vaccine antigen, NadA has been shown to induce high levels of bactericidal antibodies; however, the domains important for protective response are still unknown. In order to further investigate its immunogenic properties, we have characterized the murine IgG1 mAb (6E3) that was able to recognize the 2 main antigenic variants of NadA on the surface of MenB strains. The epitope targeted by mAb 6E3 was mapped by hydrogen-deuterium exchange mass spectrometry and shown to be located on the coiled-coil stalk region of NadA (aa 206-249). Although no serum bactericidal activity was observed for murine IgG1 mAb 6E3, functional activity was restored when using chimeric antibodies in which the variable regions of the murine mAb 6E3 were fused to human IgG3 constant regions, thus confirming the protective nature of the mAb 6E3 epitope. The use of chimeric antibody molecules will enable future investigations of complement-mediated antibody functionality independently of the Fc-mediated differences in complement activation.
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http://dx.doi.org/10.1096/fj.15-273813DOI Listing
January 2016

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

Neisseria adhesin A variation and revised nomenclature scheme.

Clin Vaccine Immunol 2014 Jul 7;21(7):966-71. Epub 2014 May 7.

Department of Zoology, University of Oxford, Oxford, United Kingdom.

Neisseria adhesin A (NadA), involved in the adhesion and invasion of Neisseria meningitidis into host tissues, is one of the major components of Bexsero, a novel multicomponent vaccine licensed for protection against meningococcal serogroup B in Europe, Australia, and Canada. NadA has been identified in approximately 30% of clinical isolates and in a much lower proportion of carrier isolates. Three protein variants were originally identified in invasive meningococci and named NadA-1, NadA-2, and NadA-3, whereas most carrier isolates either lacked the gene or harbored a different variant, NadA-4. Further analysis of isolates belonging to the sequence type 213 (ST-213) clonal complex identified NadA-5, which was structurally similar to NadA-4, but more distantly related to NadA-1, -2, and -3. At the time of this writing, more than 89 distinct nadA allele sequences and 43 distinct peptides have been described. Here, we present a revised nomenclature system, taking into account the complete data set, which is compatible with previous classification schemes and is expandable. The main features of this new scheme include (i) the grouping of the previously named NadA-2 and NadA-3 variants into a single NadA-2/3 variant, (ii) the grouping of the previously assigned NadA-4 and NadA-5 variants into a single NadA-4/5 variant, (iii) the introduction of an additional variant (NadA-6), and (iv) the classification of the variants into two main groups, named groups I and II. To facilitate querying of the sequences and submission of new allele sequences, the nucleotide and amino acid sequences are available at http://pubmlst.org/neisseria/NadA/.
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http://dx.doi.org/10.1128/CVI.00825-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4097447PMC
July 2014

Immune responses to pneumococcal pilus RrgA and RrgB antigens and their relationship with pneumococcal carriage in humans.

J Infect 2014 Jun 6;68(6):562-71. Epub 2014 Feb 6.

Department of Clinical Infection, Microbiology and Immunology, Institute of Infection and Global Health, University of Liverpool, UK. Electronic address:

Objectives: Pneumococcal pilus antigens are shown to be important in pneumococcal pathogenesis and induce protective immunity in animal studies, but data in humans are limited. We aimed to investigate serum and mucosal immune responses to pilus-1 proteins (RrgA and RrgB) and their relationship with pneumococcal carriage in humans.

Methods: Serum and salivary antibodies to RrgA and RrgB in children and adults were analysed by ELISA and immunoblotting. Induction of B cell antibody responses to RrgA and RrgB in nasopharynx-associated lymphoid tissue was studied by ELISpot assay following stimulation with pneumococcal culture supernatants containing pilus proteins.

Results: Significant levels of serum anti-RrgA and -RrgB antibodies were observed, and anti-RrgA antibody appeared to develop earlier in childhood. Importantly, anti-RrgA IgG titres in both serum and saliva were shown to be higher in culture-negative children than in those who were culture-positive for Streptococcus pneumoniae. Stimulation of adenotonsillar cells with pneumococcal culture supernatant induced significant RrgA- and RrgB-specific antibody secreting cells and antibody production.

Conclusions: Pneumococcal pilus antigens, particularly RrgA, seem to induce significant serum and mucosal antibody responses that may contribute to natural immunity against pneumococcal carriage in children.
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http://dx.doi.org/10.1016/j.jinf.2014.01.013DOI Listing
June 2014

Variation of pneumococcal Pilus-1 expression results in vaccine escape during Experimental Otitis Media [EOM].

PLoS One 2014 8;9(1):e83798. Epub 2014 Jan 8.

Section of Pediatric Infectious Diseases, Maxwell Finland Laboratory for Infectious Diseases, Boston Medical Center, Boston, Massachusetts, United States of America.

Unlabelled: The pneumococcal Pilus-1 enhances attachment to epithelial cells in the respiratory tract and subsequent invasion. Pilus-1 expression is bi-stable and positively regulated by the RlrA transcriptional regulator. To delineate the role of pilus-1 in Experimental Otitis Media (EOM), we evaluated colonization and disease due to a Streptococcus pneumoniae (SP) wild type strain (Taiwan19F-14 wt) and its otherwise isogenic pilus-1 and pilus-2 deficient mutant (Taiwan19F-14 ΔPI-1/PI-2-) as well as potential for a chimeric protein (RrgB321) vaccine candidate for prevention of middle ear (ME) disease.

Methods: Chinchillas were challenged intranasally with either Taiwan19F-14 wt or Taiwan19F-14PI-1/PI-2 deficient mutant. ME status was assessed and direct cultures performed. New cohorts of animals were immunized with RrgB321 or alum. Intranasal challenge with Taiwan19F-14 wt [erythromycin susceptible E(S)] was performed. Subsequently, a second cohort of animals was immunized and challenged with either Taiwan19F-14 wt or a Pilus-1 over-expressing mutant [Taiwan19F-14+pMU1328_Pc-rlrA mutant; E resistant (R)] strain. Pilus-1 expression was analyzed in SP isolated from nasopharynx (NP) and ME fluids by flow cytometry.

Results: Culture positive EOM developed following challenge with either wild type SP (Taiwan19F-14) or its pilus-1 deficient mutant. Culture positive EOM developed following challenge with wild type in both RrgB321 immunized and control animals. Pilus-1 expression in ME fluids was significantly higher in controls compared to immunized chinchillas. In second cohort of immunized and control animals challenged with the over-expressing Pilus-1 mutant, delayed development of EOM in the immunized animals was observed. Pneumococci recovered from ME fluid of immunized animals were no longer E(R) signifying the loss of the pMU1328_Pc-rlrA plasmid.

Conclusion: Pneumococcal pilus-1 was not essential for EOM. Regulation of Pilus-1 expression in ME fluids in the presence of anti RrgB321 antibody was essential for survival of S. pneumoniae. Pneumococci have evolved mechanisms of regulation of non-essential surface proteins to evade host defenses.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0083798PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3885439PMC
September 2014

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

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

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

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

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

An extended multi-locus molecular typing schema for Streptococcus pneumoniae demonstrates that a limited number of capsular switch events is responsible for serotype heterogeneity of closely related strains from different countries.

Infect Genet Evol 2013 Jan 27;13:151-61. Epub 2012 Sep 27.

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

Streptococcus pneumoniae is a major cause of morbidity and mortality worldwide. Pneumococcal strains are classified according to their capsular serotype and through a Multi-Locus Sequence Typing schema (MLST) based on the sequencing of seven housekeeping genes. However, strains with a defined allelic profile (Sequence Type, ST) can have different serotypes, suggesting that the micro-evolution of the MLST lineages leads to a considerable degree of phenotypic variability. To better investigate the genetic diversity within these lineages, we set-up and then validated an extended molecular typing schema (96-MLST) based on the sequencing of ninety-six genomic loci. 96-MLST loci were designed within core-genes in a collection of 39 complete genomes of S. pneumoniae. None of the capsular genes was included in the schema. When tested on a collection of 69 isolates, 96-MLST was able to partition strains with the same ST and diverse serotypes into groups that were homogenous for capsular serotype, improving our understanding of the evolution of epidemiologically relevant lineages. Phylogenetic sequence analysis showed that the capsular heterogeneity of three STs that were sampled more extensively could be traced back to a limited number of capsular switch events, indicating that changes of serotype occur occasionally during the short term expansion of clones. Moreover, a geographical structure of ST156 was identified, suggesting that the resolution guaranteed by this method is sufficient for phylogeographic studies. In conclusion, we showed that an extended typing schema was able to characterize the expansion of individual lineages in a complex species such as S. pneumoniae.
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http://dx.doi.org/10.1016/j.meegid.2012.09.008DOI Listing
January 2013

Immunization with the RrgB321 fusion protein protects mice against both high and low pilus-expressing Streptococcus pneumoniae populations.

Vaccine 2012 Feb 30;30(7):1349-56. Epub 2011 Dec 30.

Novartis Vaccines and Diagnostics, 53100 Siena, Italy.

RrgB321, a fusion protein of the three Streptococcus pneumoniae pilus-1 backbone RrgB variants, is protective in vivo against pilus islet 1 (PI-1) positive pneumococci. In addition, antibodies to RrgB321 mediate a complement-dependent opsonophagocytosis of PI-1 positive strains at levels comparable to those obtained with antisera against glycoconjugate vaccines. In the pneumococcus, pilus-1 displays a biphasic expression pattern, with different proportions of two bacterial phenotypes, one expressing and one not expressing the pilus-1. These two populations can be stably separated in vitro giving rise to the enriched high (H) and low (L) pilus expressing populations. In this work we demonstrate that: (i) the opsonophagocytic killing mediated in vitro by RrgB321 antisera is strictly dependent on the pilus expression ratio of the strain used; (ii) during the opsonophagocytosis assay pilus-expressing pneumococci are selectively killed, and (iii) no switch towards the pilus non-expressing phenotype can be observed. Furthermore, in sepsis and pneumonia models, mice immunized with RrgB321 are significantly protected against challenge with either the H or the L pilus-expressing population of strains representative of the three RrgB variants. This suggests that the pilus-1 expression is not down-regulated, and also that the expression of the pilus-1 could be up-regulated in vivo. In conclusion, these data provide evidence that RrgB321 is protective against PI-1 positive strains regardless of their pilus expression level, and support the rationale for the inclusion of this fusion protein into a multi-component protein-based pneumococcal vaccine.
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http://dx.doi.org/10.1016/j.vaccine.2011.12.080DOI Listing
February 2012

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

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

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

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

The Streptococcus pneumoniae pilus-1 displays a biphasic expression pattern.

PLoS One 2011 22;6(6):e21269. Epub 2011 Jun 22.

Novartis Vaccines and Diagnostics, Siena, Italy.

The Streptococcus pneumoniae pilus-1 is encoded by pilus islet 1 (PI-1), which has three clonal variants (clade I, II and III) and is present in about 30% of clinical pneumococcal isolates. In vitro and in vivo assays have demonstrated that pilus-1 is involved in attachment to epithelial cells and virulence, as well as protection in mouse models of infection. Several reports suggest that pilus-1 expression is tightly regulated and involves the interplay of numerous genetic regulators, including the PI-1 positive regulator RlrA. In this report we provide evidence that pilus expression, when analyzed at the single-cell level in PI-1 positive strains, is biphasic. In fact, the strains present two phenotypically different sub-populations of bacteria, one that expresses the pilus, while the other does not. The proportions of these two phenotypes are variable among the strains tested and are not influenced by genotype, serotype, growth conditions, colony morphology or by the presence of antibodies directed toward the pilus components. Two sub-populations, enriched in pilus expressing or not expressing bacteria were obtained by means of colony selection and immuno-detection methods for five strains. PI-1 sequencing in the two sub-populations revealed the absence of mutations, thus indicating that the biphasic expression observed is not due to a genetic modification within PI-1. Microarray expression profile and western blot analyses on whole bacterial lysates performed comparing the two enriched sub-populations, revealed that pilus expression is regulated at the transcriptional level (on/off regulation), and that there are no other genes, in addition to those encoded by PI-1, concurrently regulated across the strains tested. Finally, we provide evidence that the over-expression of the RrlA positive regulator is sufficient to induce pilus expression in pilus-1 negative bacteria. Overall, the data presented here suggest that the observed biphasic pilus expression phenotype could be an example of bistability in pneumococcus.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0021269PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3120856PMC
November 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

Structure and dynamics of the pan-genome of Streptococcus pneumoniae and closely related species.

Genome Biol 2010 29;11(10):R107. Epub 2010 Oct 29.

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

Background: Streptococcus pneumoniae is one of the most important causes of microbial diseases in humans. The genomes of 44 diverse strains of S. pneumoniae were analyzed and compared with strains of non-pathogenic streptococci of the Mitis group.

Results: Despite evidence of extensive recombination, the S. pneumoniae phylogenetic tree revealed six major lineages. With the exception of serotype 1, the tree correlated poorly with capsular serotype, geographical site of isolation and disease outcome. The distribution of dispensable genes--genes present in more than one strain but not in all strains--was consistent with phylogeny, although horizontal gene transfer events attenuated this correlation in the case of ancient lineages. Homologous recombination, involving short stretches of DNA, was the dominant evolutionary process of the core genome of S. pneumoniae. Genetic exchange occurred both within and across the borders of the species, and S. mitis was the main reservoir of genetic diversity of S. pneumoniae. The pan-genome size of S. pneumoniae increased logarithmically with the number of strains and linearly with the number of polymorphic sites of the sampled genomes, suggesting that acquired genes accumulate proportionately to the age of clones. Most genes associated with pathogenicity were shared by all S. pneumoniae strains, but were also present in S. mitis, S. oralis and S. infantis, indicating that these genes are not sufficient to determine virulence.

Conclusions: Genetic exchange with related species sharing the same ecological niche is the main mechanism of evolution of S. pneumoniae. The open pan-genome guarantees the species a quick and economical response to diverse environments.
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http://dx.doi.org/10.1186/gb-2010-11-10-r107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3218663PMC
June 2011