Publications by authors named "Matthew J Bottomley"

43 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

Structure, dynamics and immunogenicity of a catalytically inactive CC chemokine-degrading protease SpyCEP from .

Comput Struct Biotechnol J 2020 13;18:650-660. Epub 2020 Mar 13.

Department of Life Sciences, Imperial College London, South Kensington Campus, SW7 2AZ, UK.

Over 18 million disease cases and half a million deaths worldwide are estimated to be caused annually by Group A Streptococcus. A vaccine to prevent GAS disease is urgently needed. SpyCEP (Streptococcus Cell-Envelope Proteinase) is a surface-exposed serine protease that inactivates chemokines, impairing neutrophil recruitment and bacterial clearance, and has shown promising immunogenicity in preclinical models. Although SpyCEP structure has been partially characterized, a more complete and higher resolution understanding of its antigenic features would be desirable prior to large scale manufacturing. To address these gaps and facilitate development of this globally important vaccine, we performed immunogenicity studies with a safety-engineered SpyCEP mutant, and comprehensively characterized its structure by combining X-ray crystallography, NMR spectroscopy and molecular dynamics simulations. We found that the catalytically-inactive SpyCEP antigen conferred protection similar to wild-type SpyCEP in a mouse infection model. Further, a new higher-resolution crystal structure of the inactive SpyCEP mutant provided new insights into this large chemokine protease comprising nine domains derived from two non-covalently linked fragments. NMR spectroscopy and molecular simulation analyses revealed conformational flexibility that is likely important for optimal substrate recognition and overall function. These combined immunogenicity and structural data demonstrate that the full-length SpyCEP inactive mutant is a strong candidate human vaccine antigen. These findings show how a multi-disciplinary study was used to overcome obstacles in the development of a GAS vaccine, an approach applicable to other future vaccine programs. Moreover, the information provided may also facilitate the structure-based discovery of small-molecule therapeutics targeting SpyCEP protease inhibition.
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http://dx.doi.org/10.1016/j.csbj.2020.03.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7113628PMC
March 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

The Role of the Immune System in Cutaneous Squamous Cell Carcinoma.

Int J Mol Sci 2019 Apr 24;20(8). Epub 2019 Apr 24.

Centre for Cell Biology and Cutaneous Research, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, London E1 2AT, UK.

Cutaneous squamous cell carcinoma (cSCC) is the second most common skin cancer. In immunosuppressed populations it is a source of considerable morbidity and mortality due to its enhanced recurrence and metastatic potential. In common with many malignancies, leucocyte populations are both protective against cancer development and also play a role in 'sculpting' the nascent tumor, leading to loss of immunogenicity and tumor progression. UV radiation and chronic viral carriage may represent unique risk factors for cSCC development, and the immune system plays a key role in modulating the response to both. In this review, we discuss the lessons learned from animal and ex vivo human studies of the role of individual leucocyte subpopulations in the development of cutaneous SCC. We then discuss the insights into cSCC immunity gleaned from studies in humans, particularly in populations receiving pharmacological immunosuppression such as transplant recipients. Similar insights in other malignancies have led to exciting and novel immune therapies, which are beginning to emerge into the cSCC clinical arena.
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http://dx.doi.org/10.3390/ijms20082009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6515307PMC
April 2019

NadA3 Structures Reveal Undecad Coiled Coils and LOX1 Binding Regions Competed by Meningococcus B Vaccine-Elicited Human Antibodies.

mBio 2018 10 16;9(5). Epub 2018 Oct 16.

GSK, Siena, Italy

serogroup B (MenB) is a major cause of sepsis and invasive meningococcal disease. A multicomponent vaccine, 4CMenB, is approved for protection against MenB. Neisserial adhesin A (NadA) is one of the main vaccine antigens, acts in host cell adhesion, and may influence colonization and invasion. Six major genetic variants of NadA exist and can be classified into immunologically distinct groups I and II. Knowledge of the crystal structure of the 4CMenB vaccine component NadA3 (group I) would improve understanding of its immunogenicity, folding, and functional properties and might aid antigen design. Here, X-ray crystallography, biochemical, and cellular studies were used to deeply characterize NadA3. The NadA3 crystal structure is reported; it revealed two unexpected regions of undecad coiled-coil motifs and other conformational differences from NadA5 (group II) not predicted by previous analyses. Structure-guided engineering was performed to increase NadA3 thermostability, and a second crystal structure confirmed the improved packing. Functional NadA3 residues mediating interactions with human receptor LOX-1 were identified. Also, for two protective vaccine-elicited human monoclonal antibodies (5D11, 12H11), we mapped key NadA3 epitopes. These vaccine-elicited human MAbs competed binding of NadA3 to LOX-1, suggesting their potential to inhibit host-pathogen colonizing interactions. The data presented provide a significant advance in the understanding of the structure, immunogenicity and function of NadA, one of the main antigens of the multicomponent meningococcus B vaccine. The bacterial microbe serogroup B (MenB) is a major cause of devastating meningococcal disease. An approved multicomponent vaccine, 4CMenB, protects against MenB. Neisserial adhesin A (NadA) is a key vaccine antigen and acts in host cell-pathogen interactions. We investigated the 4CMenB vaccine component NadA3 in order to improve the understanding of its immunogenicity, structure, and function and to aid antigen design. We report crystal structures of NadA3, revealing unexpected structural motifs, and other conformational differences from the NadA5 orthologue studied previously. We performed structure-based antigen design to engineer increased NadA3 thermostability. Functional NadA3 residues mediating interactions with the human receptor LOX-1 and vaccine-elicited human antibodies were identified. These antibodies competed binding of NadA3 to LOX-1, suggesting their potential to inhibit host-pathogen colonizing interactions. Our data provide a significant advance in the overall understanding of the 4CMenB vaccine antigen NadA.
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http://dx.doi.org/10.1128/mBio.01914-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6191539PMC
October 2018

Non-Enzymatic and Site-Specific Glycan Shedding: A Novel Protein Degradation Pathway Observed in a Stabilized Form of RSV Prefusion F Protein.

Anal Chem 2018 09 7;90(18):10897-10902. Epub 2018 Sep 7.

Analytical R&D , Slaoui Center for Vaccines Research, GSK Vaccines , 14200 Shady Grove Road , Rockville , Maryland 20850 , United States.

Stability is one of the critical attributes of a protein-based therapeutic or vaccine product, which is directly linked to product quality and efficacy. Elucidating protein degradation pathways is required to obtain thorough understanding of the product and ensure degradation products are properly monitored. We observed a unique protein degradation involving nonenzyme catalyzed loss of a complete N-linked glycan under stress condition from an engineered respiratory syncytial virus (RSV) prefusion F protein (RSVPreF3). Investigations involving mass spectrometry, molecular modeling, and mutagenesis revealed that the glycan shedding was site-specific, dependent on structural elements, and required a glycine residue immediately following the site of glycosylation. The glycan loss did not negatively affect the binding between the main immunogenic epitope Site Ø and the neutralizing antibody D25. Further study indicated that the glycan shedding followed a similar but different mechanism than that of conventional deamidation. Since glycosylation is an important attribute for many recombinant therapeutic proteins or vaccine antigens, the finding from this study suggests the need to monitor this new type of degradation, especially when glycosylation has an impact on efficacy or safety.
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http://dx.doi.org/10.1021/acs.analchem.8b02402DOI Listing
September 2018

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

Crystal structure reveals vaccine elicited bactericidal human antibody targeting a conserved epitope on meningococcal fHbp.

Nat Commun 2018 02 6;9(1):528. Epub 2018 Feb 6.

GSK Vaccines, 14200 Shady Grove Road, Rockville, MD, 20817, USA.

Data obtained recently in the United Kingdom following a nationwide infant immunization program against serogroup B Neisseria meningitidis (MenB) reported >80% 4CMenB vaccine-mediated protection. Factor H-binding protein (fHbp) is a meningococcal virulence factor and a component of two new MenB vaccines. Here, we investigated the structural bases underlying the fHbp-dependent protective antibody response in humans, which might inform future antigen design efforts. We present the co-crystal structure of a human antibody Fab targeting fHbp. The vaccine-elicited Fab 1A12 is cross-reactive and targets an epitope highly conserved across the repertoire of three naturally occurring fHbp variants. The free Fab structure highlights conformational rearrangements occurring upon antigen binding. Importantly, 1A12 is bactericidal against MenB strains expressing fHbp from all three variants. Our results reveal important immunological features potentially contributing to the broad protection conferred by fHbp vaccination. Our studies fuel the rationale of presenting conserved protein epitopes when developing broadly protective vaccines.
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http://dx.doi.org/10.1038/s41467-018-02827-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5802752PMC
February 2018

Impact of intrapatient variability (IPV) in tacrolimus trough levels on long-term renal transplant function: multicentre collaborative retrospective cohort study protocol.

BMJ Open 2017 Jul 28;7(7):e016144. Epub 2017 Jul 28.

Department of Transplantation, NHS Lothian, Edinburgh, UK.

Introduction: High intrapatient variability (IPV) in tacrolimus trough levels has been shown to be associated with higher rates of renal transplant failure. There is no consensus on what level of IPV constitutes a risk of graft loss. The establishment of such a threshold could help to guide clinicians in identifying at-risk patients to receive targeted interventions to improve IPV and thus outcomes.

Methods And Analysis: A multicentre Transplant Audit Collaborative has been established to conduct a retrospective study examining tacrolimus IPV and renal transplant outcomes. Patients in receipt of a renal transplant at participating centres between 2009 and 2014 and fulfilling the inclusion criteria will be included in the study. The aim is to recruit a minimum of 1600 patients with follow-up spanning at least 2 years in order to determine a threshold IPV above which a renal transplant recipient would be considered at increased risk of graft loss. The study also aims to determine any national or regional trends in IPV and any demographic associations.

Ethics And Dissemination: Consent will not be sought from patients whose data are used in this study as no additional procedures or information will be required from participants beyond that which would normally take place as part of clinical care. The study will be registered locally in each participating centre in line with local research and development protocols. It is anticipated that the results of this audit will be disseminated locally, in participating NHS Trusts, through national and international meetings and publications in peer-reviewed journals.
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http://dx.doi.org/10.1136/bmjopen-2017-016144DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5642769PMC
July 2017

Application of Operational Tolerance Signatures Are Limited by Variability and Type of Immunosuppression in Renal Transplant Recipients: A Cross-Sectional Study.

Transplant Direct 2017 Jan 21;3(1):e125. Epub 2016 Dec 21.

Nuffield Department of Surgical Sciences, University of Oxford, Oxford, United Kingdom.

Background: Renal transplant recipients (RTR) frequently develop complications relating to chronic immunosuppression. Identifying RTR who could safely reduce immunosuppression is therefore highly desirable. We hypothesized that "signatures" described in RTR who have stopped immunosuppression but maintained stable graft function ("operational tolerance") may enable identification of immunosuppressed RTR who are candidates for immunosuppression minimization. However, the effect of immunosuppression itself on these signatures and circulating B-cell populations is currently unknown.

Methods: We undertook a cross-sectional study of 117 RTR to assess the effect of immunosuppression upon circulating B cell populations, humoral alloresponse and 2 previously published "signatures" of operational tolerance.

Results: Immunosuppression associated with alterations in both published "signatures." Azathioprine associated with a decrease in transitional and naive B-cell numbers and calcineurin inhibition associated with an increase in the number of circulating plasmablasts. However, only azathioprine use associated with the presence of donor-specific anti-HLA IgG antibodies. Calcineurin inhibition associated with an increase in total serum IgM but not IgG. Data were corrected for age, time since last transplant, and other immunosuppression.

Conclusions: Current signatures of operational tolerance may be significantly affected by immunosuppressive regimen, which may hinder use in their current form in clinical practice. Calcineurin inhibition may prevent the development of long-lasting humoral alloresponses, whereas azathioprine therapy may be associated with donor specific antibody development.
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http://dx.doi.org/10.1097/TXD.0000000000000638DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5361564PMC
January 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

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

Molecular Basis of Ligand-Dependent Regulation of NadR, the Transcriptional Repressor of Meningococcal Virulence Factor NadA.

PLoS Pathog 2016 04 22;12(4):e1005557. Epub 2016 Apr 22.

GSK Vaccines Srl, Siena, Italy.

Neisseria adhesin A (NadA) is present on the meningococcal surface and contributes to adhesion to and invasion of human cells. NadA is also one of three recombinant antigens in the recently-approved Bexsero vaccine, which protects against serogroup B meningococcus. The amount of NadA on the bacterial surface is of direct relevance in the constant battle of host-pathogen interactions: it influences the ability of the pathogen to engage human cell surface-exposed receptors and, conversely, the bacterial susceptibility to the antibody-mediated immune response. It is therefore important to understand the mechanisms which regulate nadA expression levels, which are predominantly controlled by the transcriptional regulator NadR (Neisseria adhesin A Regulator) both in vitro and in vivo. NadR binds the nadA promoter and represses gene transcription. In the presence of 4-hydroxyphenylacetate (4-HPA), a catabolite present in human saliva both under physiological conditions and during bacterial infection, the binding of NadR to the nadA promoter is attenuated and nadA expression is induced. NadR also mediates ligand-dependent regulation of many other meningococcal genes, for example the highly-conserved multiple adhesin family (maf) genes, which encode proteins emerging with important roles in host-pathogen interactions, immune evasion and niche adaptation. To gain insights into the regulation of NadR mediated by 4-HPA, we combined structural, biochemical, and mutagenesis studies. In particular, two new crystal structures of ligand-free and ligand-bound NadR revealed (i) the molecular basis of 'conformational selection' by which a single molecule of 4-HPA binds and stabilizes dimeric NadR in a conformation unsuitable for DNA-binding, (ii) molecular explanations for the binding specificities of different hydroxyphenylacetate ligands, including 3Cl,4-HPA which is produced during inflammation, (iii) the presence of a leucine residue essential for dimerization and conserved in many MarR family proteins, and (iv) four residues (His7, Ser9, Asn11 and Phe25), which are involved in binding 4-HPA, and were confirmed in vitro to have key roles in the regulatory mechanism in bacteria. Overall, this study deepens our molecular understanding of the sophisticated regulatory mechanisms of the expression of nadA and other genes governed by NadR, dependent on interactions with niche-specific signal molecules that may play important roles during meningococcal pathogenesis.
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http://dx.doi.org/10.1371/journal.ppat.1005557DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4841544PMC
April 2016

Reverse vaccinology 2.0: Human immunology instructs vaccine antigen design.

J Exp Med 2016 Apr 28;213(4):469-81. Epub 2016 Mar 28.

GlaxoSmithKline Vaccines S.r.l., 53100 Siena, Italy.

Traditionally, vaccines have been developed by cultivating infectious agents and isolating the inactivated whole pathogen or some of its purified components. 20 years ago, reverse vaccinology enabled vaccine discovery and design based on information deriving from the sequence of microbial genomes rather than via the growth of pathogens. Today, the high throughput discovery of protective human antibodies, sequencing of the B cell repertoire, and the increasing structural characterization of protective antigens and epitopes provide the molecular and mechanistic understanding to drive the discovery of novel vaccines that were previously impossible. We are entering a "reverse vaccinology 2.0" era.
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http://dx.doi.org/10.1084/jem.20151960DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4821650PMC
April 2016

Self-assembling protein nanoparticles in the design of vaccines.

Comput Struct Biotechnol J 2016 26;14:58-68. Epub 2015 Nov 26.

GlaxoSmithKline Vaccines S.r.l., Via Fiorentina 1, 53100 Siena, Italy.

For over 100 years, vaccines have been one of the most effective medical interventions for reducing infectious disease, and are estimated to save millions of lives globally each year. Nevertheless, many diseases are not yet preventable by vaccination. This large unmet medical need demands further research and the development of novel vaccines with high efficacy and safety. Compared to the 19th and early 20th century vaccines that were made of killed, inactivated, or live-attenuated pathogens, modern vaccines containing isolated, highly purified antigenic protein subunits are safer but tend to induce lower levels of protective immunity. One strategy to overcome the latter is to design antigen nanoparticles: assemblies of polypeptides that present multiple copies of subunit antigens in well-ordered arrays with defined orientations that can potentially mimic the repetitiveness, geometry, size, and shape of the natural host-pathogen surface interactions. Such nanoparticles offer a collective strength of multiple binding sites (avidity) and can provide improved antigen stability and immunogenicity. Several exciting advances have emerged lately, including preclinical evidence that this strategy may be applicable for the development of innovative new vaccines, for example, protecting against influenza, human immunodeficiency virus, and respiratory syncytial virus. Here, we provide a concise review of a critical selection of data that demonstrate the potential of this field. In addition, we highlight how the use of self-assembling protein nanoparticles can be effectively combined with the emerging discipline of structural vaccinology for maximum impact in the rational design of vaccine antigens.
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http://dx.doi.org/10.1016/j.csbj.2015.11.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4706605PMC
February 2016

CD8+ Immunosenescence Predicts Post-Transplant Cutaneous Squamous Cell Carcinoma in High-Risk Patients.

J Am Soc Nephrol 2016 05 12;27(5):1505-15. Epub 2015 Nov 12.

Transplantation Research Immunology Group, Nuffield Department of Surgical Sciences, University of Oxford, Oxford, United Kingdom; and

Most morbidity associated with malignancy in long-term renal transplant recipients is due to cutaneous squamous cell carcinoma (SCC). Previously identified measures to stratify SCC risk have limited use, however. We hypothesized that an increased proportion of senescent, terminally differentiated CD8(+) T cells would identify renal transplant recipients at elevated SCC risk. Peripheral blood lymphocytes were isolated from 117 stable transplant recipients at high risk of SCC and analyzed phenotypically by flow cytometry. Participants were followed up prospectively for SCC development. The predictive value of variables was assessed using Cox regression. Age at transplant and enrollment, dialysis duration, and previous disease were predictive of SCC development during follow-up. Previously published clinical phenotype-based risk scores lost predictive value with the removal of age as a covariate. The percentage of CD57-expressing CD8(+) T cells was the strongest immunologic predictor of future SCC and correlated with increasing CD8(+) T cell differentiation. We dichotomized participants into those with a majority (CD57hi) and a minority (CD57lo) of CD8(+) T cells expressing CD57; CD57hi participants were more likely to develop SCC during follow-up (hazard ratio, 2.9; 95% confidence interval, 1.0 to 8.0), independent of potential confounders, and tended to develop earlier recurrence. The CD57hi phenotype was stable with time and associated with increasing age and cytomegalovirus seropositivity. Our results show that the CD57hi phenotype is a strong predictor of SCC development and recurrence in this cohort of long-term, high-risk renal transplant recipients. This information may allow identification of recipients who may benefit from intensive dermatologic screening and immunosuppression reduction.
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http://dx.doi.org/10.1681/ASN.2015030250DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4849821PMC
May 2016

An observational study of patient characteristics and mortality following hypoglycemia in the community.

BMJ Open Diabetes Res Care 2015 30;3(1):e000094. Epub 2015 Jun 30.

Division of Cardiovascular and Diabetes Research , Leeds Institute of Cardiovascular and Metabolic Medicine (LICAMM), The University of Leeds , Leeds , UK ; The Leeds Teaching Hospitals Trust, St James' University Hospital , Leeds , UK.

Objectives: Characterize patients with diabetes with severe hypoglycemia requiring emergency services intervention at home and investigate 12-month mortality.

Research Design And Methods: Emergency services call-outs for hypoglycemia were recorded between 2005 and 2013 in an area covering 34 000 patients with diabetes. Patient characteristics were documented together with capillary blood glucose (CBG), glycated hemoglobin (HbA1c), and treatment for hypoglycemia; 12-month mortality and variables influencing survival were analyzed.

Results: In 1835 episodes among 1156 patients, 45% had type 1 diabetes (68.2% males) and 44% had type 2 diabetes (49.4% males), with a minority unclassified. CBG at presentation (mean±SD) was 1.76±0.72 mmol/L in patients with type 1 diabetes and 1.96±0.68 mmol/L in patients with type 2 diabetes (p<0.0001), with a higher HbA1c in the former group (8.3±1.52% (67.5±16.4 mmol/mol) and 7.8±1.74% (61.6±19.0 mmol/mol), respectively; p<0.0001). A third of patients with type 2 diabetes were not on insulin therapy and displayed lower HbA1c compared with insulin users. Glucagon was used in 37% of patients with type 1 diabetes and 28% of patients with type 2 diabetes (p<0.0001). One-year mortality was 4.45% in type 1 diabetes and 22.1% in type 2 diabetes. Age and type of diabetes were predictive of mortality in multivariable analysis, whereas CBG levels/frequency of hypoglycemia had no effect.

Conclusions: Severe hypoglycemia in the community is common with a male predominance in type 1 diabetes. Severe hypoglycemia in non-insulin treated patients with type 2 diabetes is associated with lower HbA1c compared with insulin users. Severe hypoglycemia appears to be associated with increased mortality at 12 months, particularly in type 2 diabetes.
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http://dx.doi.org/10.1136/bmjdrc-2015-000094DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4486683PMC
July 2015

Protein Crystallography in Vaccine Research and Development.

Int J Mol Sci 2015 Jun 9;16(6):13106-40. Epub 2015 Jun 9.

Protein Biochemistry Department, Novartis Vaccines & Diagnostics s.r.l. (a GSK Company), Via Fiorentina 1, 53100 Siena, Italy.

The use of protein X-ray crystallography for structure-based design of small-molecule drugs is well-documented and includes several notable success stories. However, it is less well-known that structural biology has emerged as a major tool for the design of novel vaccine antigens. Here, we review the important contributions that protein crystallography has made so far to vaccine research and development. We discuss several examples of the crystallographic characterization of vaccine antigen structures, alone or in complexes with ligands or receptors. We cover the critical role of high-resolution epitope mapping by reviewing structures of complexes between antigens and their cognate neutralizing, or protective, antibody fragments. Most importantly, we provide recent examples where structural insights obtained via protein crystallography have been used to design novel optimized vaccine antigens. This review aims to illustrate the value of protein crystallography in the emerging discipline of structural vaccinology and its impact on the rational design of vaccines.
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http://dx.doi.org/10.3390/ijms160613106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4490488PMC
June 2015

Structure and assembly of group B streptococcus pilus 2b backbone protein.

PLoS One 2015 5;10(5):e0125875. Epub 2015 May 5.

Novartis Vaccines and Diagnostics, Siena, Italy.

Group B Streptococcus (GBS) is a major cause of invasive disease in infants. Like other Gram-positive bacteria, GBS uses a sortase C-catalyzed transpeptidation mechanism to generate cell surface pili from backbone and ancillary pilin precursor substrates. The three pilus types identified in GBS contain structural subunits that are highly immunogenic and are promising candidates for the development of a broadly-protective vaccine. Here we report the X-ray crystal structure of the backbone protein of pilus 2b (BP-2b) at 1.06Å resolution. The structure reveals a classical IgG-like fold typical of the pilin subunits of other Gram-positive bacteria. The crystallized portion of the protein (residues 185-468) encompasses domains D2 and D3 that together confer high stability to the protein due to the presence of an internal isopeptide bond within each domain. The D2+D3 region, lacking the N-terminal D1 domain, was as potent as the entire protein in conferring protection against GBS challenge in a well-established mouse model. By site-directed mutagenesis and complementation studies in GBS knock-out strains we identified the residues and motives essential for assembly of the BP-2b monomers into high-molecular weight complexes, thus providing new insights into pilus 2b polymerization.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0125875PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4420484PMC
February 2016

Apo, Zn2+-bound and Mn2+-bound structures reveal ligand-binding properties of SitA from the pathogen Staphylococcus pseudintermedius.

Biosci Rep 2014 Nov 24;34(6):e00154. Epub 2014 Nov 24.

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

The Gram-positive bacterium Staphylococcus pseudintermedius is a leading cause of canine bacterial pyoderma, resulting in worldwide morbidity in dogs. S. pseudintermedius also causes life-threatening human infections. Furthermore, methicillin-resistant S. pseudintermedius is emerging, resembling the human health threat of methicillin-resistant Staphylococcus aureus. Therefore it is increasingly important to characterize targets for intervention strategies to counteract S. pseudintermedius infections. Here we used biophysical methods, mutagenesis, and X-ray crystallography, to define the ligand-binding properties and structure of SitA, an S. pseudintermedius surface lipoprotein. SitA was strongly and specifically stabilized by Mn2+ and Zn2+ ions. Crystal structures of SitA complexed with Mn2+ and Zn2+ revealed a canonical class III solute-binding protein with the metal cation bound in a cavity between N- and C-terminal lobes. Unexpectedly, one crystal contained both apo- and holo-forms of SitA, revealing a large side-chain reorientation of His64, and associated structural differences accompanying ligand binding. Such conformational changes may regulate fruitful engagement of the cognate ABC (ATP-binding cassette) transporter system (SitBC) required for metal uptake. These results provide the first detailed characterization and mechanistic insights for a potential therapeutic target of the major canine pathogen S. pseudintermedius, and also shed light on homologous structures in related staphylococcal pathogens afflicting humans.
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http://dx.doi.org/10.1042/BSR20140088DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4242081PMC
November 2014

Epidemiology of Group B Streptococcus in developing countries.

Vaccine 2013 Aug;31 Suppl 4:D43-5

School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India.

Group B Streptococcus (GBS) causes pneumonia, meningitis and sepsis in neonates. The current distribution pattern of GBS serotypes in developing countries such as India, China and Brazil is not clear. In order to appropriately plan for vaccination programs to address the burden of this disease in these countries, prospective population based studies are urgently needed. In our discussions, we focused on India, China and Brazil because of the membership of our workgroup, but data on other countries are also presented here. Further studies in developing countries are needed so as to better formulate appropriate public health interventions.
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http://dx.doi.org/10.1016/j.vaccine.2013.05.094DOI Listing
August 2013

Mining the bacterial unknown proteome: identification and characterization of a novel family of highly conserved protective antigens in Staphylococcus aureus.

Biochem J 2013 Nov;455(3):273-84

*Research Department, Novartis Vaccines and Diagnostics, 53100 Siena, Italy.

In the human pathogen Staphylococcus aureus, there exists an enormous diversity of proteins containing DUFs (domains of unknown function). In the present study, we characterized the family of conserved staphylococcal antigens (Csa) classified as DUF576 and taxonomically restricted to Staphylococci. The 18 Csa paralogues in S. aureus Newman are highly similar at the sequence level, yet were found to be expressed in multiple cellular locations. Extracellular Csa1A was shown to be post-translationally processed and released. Molecular interaction studies revealed that Csa1A interacts with other Csa paralogues, suggesting that these proteins are involved in the same cellular process. The structures of Csa1A and Csa1B were determined by X-ray crystallography, unveiling a peculiar structure with limited structural similarity to other known proteins. Our results provide the first detailed biological characterization of this family and confirm the uniqueness of this family also at the structural level. We also provide evidence that Csa family members elicit protective immunity in in vivo animal models of staphylococcal infections, indicating a possible important role for these proteins in S. aureus biology and pathogenesis. These findings identify the Csa family as new potential vaccine candidates, and underline the importance of mining the bacterial unknown proteome to identify new targets for preventive vaccines.
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http://dx.doi.org/10.1042/BJ20130540DOI Listing
November 2013

Update on the long-term complications of renal transplantation.

Br Med Bull 2013 2;106:117-34. Epub 2013 May 2.

Churchill Hospital, Roosevelt Drive, Oxford, UK.

Introduction: Powerful immunosuppressive regimens have reduced rejection risk, leading to an expanding cohort of long-term kidney transplant recipients who are likely to encounter practitioners in other specialties.

Sources Of Data: Key review papers and primary literature identified through searches of PubMed, Google Scholar and Medline.

Areas Of Agreement: Death from cardiovascular disease and malignancy remain the chief causes of transplant loss. Risk factors and phenotypes for these differ from the general population.

Areas Of Controversy: Many guidelines for renal transplant recipients are based on extrapolation from studies on non-transplant cohorts and may not be appropriate. Emerging studies demonstrate that established interventions in the general population are less efficacious in transplant recipients.

Growing Points: The influence of immunosuppression on the development of complications.

Areas Timely For Developing Research: Markers to guide individualized optimal immunosuppression and predict the development of complications would allow for targeted early intervention.
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http://dx.doi.org/10.1093/bmb/ldt012DOI Listing
December 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

Structural basis for group B streptococcus pilus 1 sortases C regulation and specificity.

PLoS One 2012 8;7(11):e49048. Epub 2012 Nov 8.

Novartis Vaccines and Diagnostics, Siena, Italy.

Gram-positive bacteria assemble pili through class C sortase enzymes specialized in polymerizing pilin subunits into covalently linked, high-molecular-weight, elongated structures. Here we report the crystal structures of two class C sortases (SrtC1 and SrtC2) from Group B Streptococcus (GBS) Pilus Island 1. The structures show that both sortases are comprised of two domains: an 8-stranded β-barrel catalytic core conserved among all sortase family members and a flexible N-terminal region made of two α-helices followed by a loop, known as the lid, which acts as a pseudo-substrate. In vitro experiments performed with recombinant SrtC enzymes lacking the N-terminal portion demonstrate that this region of the enzyme is dispensable for catalysis but may have key roles in substrate specificity and regulation. Moreover, in vitro FRET-based assays show that the LPXTG motif common to many sortase substrates is not the sole determinant of sortase C specificity during pilin protein recognition.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0049048PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3493515PMC
July 2013

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

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

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

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

The structure of Clostridium difficile toxin A glucosyltransferase domain bound to Mn2+ and UDP provides insights into glucosyltransferase activity and product release.

FEBS J 2012 Sep 23;279(17):3085-97. Epub 2012 Jul 23.

Novartis Vaccines and Diagnostics, Siena, Italy.

Clostridiumdifficile toxin A (TcdA) is a member of the large clostridial toxin family, and is responsible, together with C. difficile toxin B (TcdB), for many clinical symptoms d ring human infections. Like other large clostridial toxins, TcdA catalyzes the glucosylation of GTPases, and is able to inactivate small GTPases within the host cell. Here, we report the crystal structures of the TcdA glucosyltransferase domain (TcdA-GT) in the apo form and in the presence of Mn(2+) and hydrolyzed UDP-glucose. These structures, together with the recently reported crystal structure of TcdA-GT bound to UDP-glucose, provide a detailed understanding of the conformational changes of TcdA that occur during the catalytic cycle. Indeed, we present a new intermediate conformation of a so-called 'lid' loop (residues 510-522 in TcdA), concomitant with the absence of glucose in the catalytic domain. The recombinant TcdA was expressed in Brevibacillus in the inactive apo form. High thermal stability of wild-type TcdA was observed only after the addition of both Mn(2+) and UDP-glucose. The glucosylhydrolase activity, which is readily restored after reconstitution with both these cofactors, was similar to that reported for TcdB. Interestingly, we found that ammonium, like K(+) , is able to activate the UDP-glucose hydrolase activities of TcdA. Consequently, the presence of ammonium in the crystallization buffer enabled us to obtain the first crystal structure of TcdA-GT bound to the hydrolysis product UDP.
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http://dx.doi.org/10.1111/j.1742-4658.2012.08688.xDOI Listing
September 2012

The new multicomponent vaccine against meningococcal serogroup B, 4CMenB: immunological, functional and structural characterization of the antigens.

Vaccine 2012 May;30 Suppl 2:B87-97

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

Neisseria meningitidis is a major cause of endemic cases and epidemics of meningitis and devastating septicemia. Although effective vaccines exist for several serogroups of pathogenic N. meningitidis, conventional vaccinology approaches have failed to provide a universal solution for serogroup B (MenB) which consequently remains an important burden of disease worldwide. The advent of whole-genome sequencing changed the approach to vaccine development, enabling the identification of potential vaccine candidates starting directly with the genomic information, with a process named reverse vaccinology. The application of reverse vaccinology to MenB allowed the identification of new protein antigens able to induce bactericidal antibodies. Three highly immunogenic antigens (fHbp, NadA and NHBA) were combined with outer membrane vesicles and formulated for human use in a multicomponent vaccine, named 4CMenB. This is the first MenB vaccine based on recombinant proteins able to elicit a robust bactericidal immune response in adults, adolescents and infants against a broad range of serogroup B isolates. This review describes the successful story of the development of the 4CMenB vaccine, with particular emphasis on the functional, immunological and structural characterization of the protein antigens included in the vaccine.
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http://dx.doi.org/10.1016/j.vaccine.2012.01.033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3360877PMC
May 2012

Future challenges in the elimination of bacterial meningitis.

Vaccine 2012 May;30 Suppl 2:B78-86

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

Despite the widespread implementation of several effective vaccines over the past few decades, bacterial meningitis caused by Streptococcus pneumoniae, Haemophilus influenzae, Neisseria meningitidis and Group B Streptococcus (GBS) still results in unacceptably high levels of human mortality and morbidity. A residual disease burden due to bacterial meningitis is also apparent due to a number of persistent or emerging pathogens, including Mycobacterium tuberculosis, Escherichia coli, Staphylococcus aureus, Salmonella spp. and Streptococcus suis. Here, we review the current status of bacterial meningitis caused by these pathogens, highlighting how past and present vaccination programs have attempted to counter these pathogens. We discuss how improved pathogen surveillance, implementation of current vaccines, and development of novel vaccines may be expected to further reduce bacterial meningitis and related diseases in the future.
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http://dx.doi.org/10.1016/j.vaccine.2011.12.099DOI Listing
May 2012

Mechanistic implications for LDL receptor degradation from the PCSK9/LDLR structure at neutral pH.

EMBO Rep 2011 Dec 1;12(12):1300-5. Epub 2011 Dec 1.

Department of Biochemistry and Molecular Biology, IRBM P. Angeletti, Via Pontina Km 30.600, Pomezia, Rome I-00040, Italy.

The protein PCSK9 (proprotein convertase subtilisin/kexin type 9) is a key regulator of low-density lipoprotein receptor (LDLR) levels and cardiovascular health. We have determined the crystal structure of LDLR bound to PCSK9 at neutral pH. The structure shows LDLR in a new extended conformation. The PCSK9 C-terminal domain is solvent exposed, enabling cofactor binding, whereas the catalytic domain and prodomain interact with LDLR epidermal growth factor(A) and β-propeller domains, respectively. Thus, PCSK9 seems to hold LDLR in an extended conformation and to interfere with conformational rearrangements required for LDLR recycling.
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http://dx.doi.org/10.1038/embor.2011.205DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3245695PMC
December 2011