Publications by authors named "Emiliano Chiarot"

19 Publications

  • Page 1 of 1

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

Protective effect of Group B Streptococcus type-III polysaccharide conjugates against maternal colonization, ascending infection and neonatal transmission in rodent models.

Sci Rep 2018 02 7;8(1):2593. Epub 2018 Feb 7.

GSK, Siena, Italy.

Group B Streptococcus (GBS) is a normal inhabitant of recto-vaginal mucosae in up to 30% of healthy women. Colonization is a major risk factor for perinatal infection which can lead to severe complications such as stillbirth and neonatal invasive disease. Intra-partum antibiotic prophylaxis in colonized women is a safe and cost-effective preventive measure against early-onset disease in the first days of life, but has no effect on late-onset manifestations or on early maternal infection. Maternal immunization with capsular polysaccharide-based vaccines shows promise for the prevention of both early-onset and late-onset neonatal infections, although ability to prevent maternal colonization and ascending infection has been less studied. Here we investigated the effect of a GBS glycoconjugate vaccine since the very early stage of maternal GBS acquisition to neonatal outcome by rodent models of vaginal colonization and ascending infection. Immunization of female mice and rats with a type III glycoconjugate reduced vaginal colonization, infection of chorioamniotic/ placental membranes and bacterial transmission to fetuses and pups. Type III specific antibodies were detected in the blood and vagina of vaccinated mothers and their offspring. The obtained data support a potential preventive effect of GBS glycoconjugate vaccines during the different stages of pregnancy.
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http://dx.doi.org/10.1038/s41598-018-20609-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5803199PMC
February 2018

Functional activity of maternal and cord antibodies elicited by an investigational group B Streptococcus trivalent glycoconjugate vaccine in pregnant women.

J Infect 2018 05 31;76(5):449-456. Epub 2018 Jan 31.

GSK + Novartis Vaccines and Diagnostics, Siena, Italy. Electronic address:

Objectives: The main aim of this exploratory study was to evaluate functional activity of antibodies elicited by a maternal Group B Streptococcus (GBS) investigational vaccine composed of capsular polysaccharides Ia, Ib, and III conjugated to genetically detoxified Diphtheria toxin CRM. The second objective was to investigate the relationship between serotype-specific IgG concentrations and functional activity in maternal and cord sera.

Methods: Maternal and cord sera collected at baseline and at delivery from vaccine and placebo recipients during a double-blind placebo-controlled Phase II study (www.clinicaltrials.gov, NCT01446289) were tested in an opsono-phagocytic bacterial killing assay. Cord sera from vaccine recipients were also passively transferred to newborn mice to investigate conferred protection against bacterial challenge.

Results: Antibody-mediated GBS phagocytic killing was significantly increased in maternal serum at delivery and in cord sera from the investigational vaccine group as compared to the placebo group. Anti-capsular IgG concentrations above 1 µg/mL mediated in vitro killing against GBS strains belonging to all three serotypes and IgG levels correlated with functional titers. Passively administered cord sera elicited a dose-dependent protective response against all GBS serotypes in the in vivo model.

Conclusions: The maternal vaccine elicited functional antibodies that were placentally transferred. Anti-capsular IgG concentrations in maternal and cord sera were predictive of functional activity and in vivo protection in the mouse model.
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http://dx.doi.org/10.1016/j.jinf.2018.01.006DOI Listing
May 2018

Analysis of Staphylococcus aureus-Infected Mice Reveals Differential Temporal and Spatial Expression Patterns of .

Infect Immun 2017 10 20;85(10). Epub 2017 Sep 20.

GSK Vaccines Srl, Siena, Italy

is an opportunistic human pathogen and a major cause of invasive infections such as bacteremia, endocarditis, pneumonia, and wound infections. FhuD2 is a staphylococcal lipoprotein involved in the uptake of iron-hydroxymate and is under the control of the iron uptake regulator Fur. This protein is part of an investigational multicomponent vaccine formulation that has shown protective efficacy in several murine models of infection. Even though expression has been shown to be upregulated in murine kidneys infected with , it is not known whether the bacterium undergoes increased iron deprivation during prolonged infection. Furthermore, different infection niches might provide different environments and levels of iron availability, resulting in different expression patterns among organs of the same host. To address these questions, we characterized the expression of the gene and confirmed Fur-dependent regulation of its expression. We further investigated its expression in mice infected with a bioluminescent reporter strain of expressing the luciferase operon under the control of the promoter. The emission of bioluminescence in different organs was followed over a 7-day time course, and quantitative real-time PCR analysis of the RNA transcribed from the endogenous gene was performed. Using this approach, we were able to show that expression was induced during infection in all organs analyzed and that differences in expression were observed at different time points and in different infected organs. Our data suggest that undergoes increased iron deprivation during the progression of infection in diverse host organs and accordingly induces dedicated iron acquisition mechanisms. Since FhuD2 plays a central role in providing the pathogen with the required iron, further knowledge of the patterns of expression during infection will be instrumental in better defining the role of this antigen in pathogenesis and as a vaccine antigen.
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http://dx.doi.org/10.1128/IAI.00270-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5607405PMC
October 2017

Immunogenicity and protective efficacy induced by self-amplifying mRNA vaccines encoding bacterial antigens.

Vaccine 2017 01 7;35(2):361-368. Epub 2016 Dec 7.

GSK Vaccines S.r.l., Via Fiorentina 1, 53100 Siena, Italy. Electronic address:

Nucleic acid vaccines represent an attractive approach to vaccination, combining the positive attributes of both viral vectors and live-attenuated vaccines, without the inherent limitations of each technology. We have developed a novel technology, the Self-Amplifying mRNA (SAM) platform, which is based on the synthesis of self-amplifying mRNA formulated and delivered as a vaccine. SAM vaccines have been shown to stimulate robust innate and adaptive immune responses in small animals and non-human primates against a variety of viral antigens, thus representing a safe and versatile tool against viral infections. To assess whether the SAM technology could be used for a broader range of targets, we investigated the immunogenicity and efficacy of SAM vaccines expressing antigens from Group A (GAS) and Group B (GBS) Streptococci, as models of bacterial pathogens. Two prototype bacterial antigens (the double-mutated GAS Streptolysin-O (SLOdm) and the GBS pilus 2a backbone protein (BP-2a)) were successfully expressed by SAM vectors. Mice immunized with both vaccines produced significant amounts of fully functional serum antibodies. The antibody responses generated by SAM vaccines were capable of conferring consistent protection in murine models of GAS and GBS infections. Inclusion of a eukaryotic secretion signal or boosting with the recombinant protein resulted in higher specific-antibody levels and protection. Our results support the concept of using SAM vaccines as potential solution for a wide range of both viral and bacterial pathogens, due to the versatility of the manufacturing processes and the broad spectrum of elicited protective immune response.
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http://dx.doi.org/10.1016/j.vaccine.2016.11.040DOI Listing
January 2017

Staphylococcus aureus-dependent septic arthritis in murine knee joints: local immune response and beneficial effects of vaccination.

Sci Rep 2016 11 30;6:38043. Epub 2016 Nov 30.

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

Staphylococcus aureus is the major cause of human septic arthritis and osteomyelitis, which deserve special attention due to their rapid evolution and resistance to treatment. The progression of the disease depends on both bacterial presence in situ and uncontrolled disruptive immune response, which is responsible for chronic disease. Articular and bone infections are often the result of blood bacteremia, with the knees and hips being the most frequently infected joints showing the worst clinical outcome. We report the development of a hematogenous model of septic arthritis in murine knees, which progresses from an acute to a chronic phase, similarly to what occurs in humans. Characterization of the local and systemic inflammatory and immune responses following bacterial infection brought to light specific signatures of disease. Immunization of mice with the vaccine formulation we have recently described (4C-Staph), induced a strong antibody response and specific CD4+ effector memory T cells, and resulted in reduced bacterial load in the knee joints, a milder general inflammatory state and protection against bacterial-mediated cellular toxicity. Possible correlates of protection are finally proposed, which might contribute to the development of an effective vaccine for human use.
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http://dx.doi.org/10.1038/srep38043DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5128924PMC
November 2016

Positive Contribution of Adjuvanted Influenza Vaccines to the Resolution of Bacterial Superinfections.

J Infect Dis 2016 06 9;213(12):1876-85. Epub 2016 Feb 9.

GSK Vaccines S.r.l., Vaccines Research Center, Siena.

Background: Most preclinical studies assess vaccine effectiveness in single-pathogen infection models. This is unrealistic given that humans are continuously exposed to different commensals and pathogens in sequential and mixed infections. Accordingly, complications from secondary bacterial infection are a leading cause of influenza-associated morbidity and mortality. New vaccination strategies are needed to control infections on simultaneous fronts.

Methods: We compared different anti-influenza vaccines for their protective potential in a model of viral infection with bacterial superinfection. Mice were immunized with H1N1/A/California/7/2009 subunit vaccines, formulated with different adjuvants inducing either T-helper type 1 (Th1) (MF59 plus CpG)-, Th1/2 (MF59)-, or Th17 (LTK63)-prone immune responses and were sequentially challenged with mouse-adapted influenza virus H1N1/A/Puerto Rico/8/1934 and Staphylococcus aureus USA300, a clonotype emerging as a leading contributor in postinfluenza pneumonia in humans.

Results: Unadjuvanted vaccine controlled single viral infection, yet mice had considerable morbidity from viral disease and bacterial superinfection. In contrast, all adjuvanted vaccines efficiently protected mice in both conditions. Interestingly, the Th1-inducing formulation was superior to Th1/2 or Th17 inducers.

Conclusions: Our studies should help us better understand how differential immunity to influenza skews immune responses toward coinfecting bacteria and discover novel modes to prevent bacterial superinfections in the lungs of persons with influenza.
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http://dx.doi.org/10.1093/infdis/jiw048DOI Listing
June 2016

A stable luciferase reporter plasmid for in vivo imaging in murine models of Staphylococcus aureus infections.

Appl Microbiol Biotechnol 2016 Apr 21;100(7):3197-206. Epub 2015 Dec 21.

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

In vivo imaging of bioluminescent bacteria permits their visualization in infected mice, allowing spatial and temporal evaluation of infection progression. Most available bioluminescent strains were obtained by integration of the luciferase genes into the bacterial chromosome, a challenging and time-consuming approach. Recently, episomal plasmids were used, which were introduced in bacteria and expressed all genes required for bioluminescence emission. However, the plasmid was progressively lost in vitro and in vivo, if bacteria were not maintained under antibiotic selective pressure. Increased stability could be obtained inserting into the plasmid backbone sequences that assured plasmid partition between daughter bacterial cells, or caused death of bacteria that had lost the plasmid. So far, no detailed analysis was performed of either plasmid stability in vivo or contribution of different stabilizing sequence types. Here we report the construction of a plasmid, which includes the Photorhabdus luminescens lux cassette expressed under the control of a Staphylococcus aureus specific gene promoter, and toxin/antitoxin (T/A) and partition sequences (Par) conferring stability and transmissibility of the plasmid. Following infection of mice with S. aureus carrying this plasmid, we demonstrated that the promoter-lux fusion was functional in vivo, that the plasmid was retained by 70-100% of bacterial cells 7 days post-infection, and that both stabilizing sequence types were required to maximize plasmid retention. These data suggest that the plasmid can be a valuable tool to study gene expression and bacterial spread in small laboratory animals infected with S. aureus or possibly other Gram-positive human pathogens.
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http://dx.doi.org/10.1007/s00253-015-7229-2DOI Listing
April 2016

MF59- and Al(OH)3-Adjuvanted Staphylococcus aureus (4C-Staph) Vaccines Induce Sustained Protective Humoral and Cellular Immune Responses, with a Critical Role for Effector CD4 T Cells at Low Antibody Titers.

Front Immunol 2015 7;6:439. Epub 2015 Sep 7.

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

Staphylococcus aureus (S. aureus) is an important opportunistic pathogen that may cause invasive life-threatening infections, like sepsis and pneumonia. Due to the increasing antibiotic resistance, the development of an effective vaccine against S. aureus is needed. Although a correlate of protection against staphylococcal diseases is not yet established, several findings suggest that both antibodies and CD4 T cells might contribute to optimal immunity. In this study, we show that adjuvanting a multivalent vaccine (4C-Staph) with MF59, an oil-in-water emulsion licensed in human vaccines, further potentiated antigen-specific IgG titers and CD4 T-cell responses compared to alum and conferred protection in the peritonitis model of S. aureus infection. Moreover, we showed that MF59- and alum-adjuvanted 4C-Staph vaccines induced persistent antigen-specific humoral and T-cell responses, and protected mice from infection up to 4 months after immunization. Furthermore, 4C-Staph formulated with MF59 was used to investigate which immune compartment is involved in vaccine-induced protection. Using CD4 T cell-depleted mice or B cell-deficient mice, we demonstrated that both T and B-cell responses contributed to 4C-Staph vaccine-mediated protective immunity. However, the role of CD4 T cells seemed more evident in the presence of low-antibody responses. This study provides preclinical data further supporting the use of the adjuvanted 4C-Staph vaccines against S. aureus diseases, and provides critical insights on the correlates of protective immunity necessary to combat this pathogen.
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http://dx.doi.org/10.3389/fimmu.2015.00439DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4561515PMC
October 2015

Antibody-mediated immunity induced by engineered Escherichia coli OMVs carrying heterologous antigens in their lumen.

J Extracell Vesicles 2014 11;3. Epub 2014 Aug 11.

Centre for Integrative Biology, University of Trento, Trento, Italy ; Novartis Vaccines and Diagnostics, Siena, Italy.

Background: Outer membrane vesicles (OMVs) from Gram-negative bacteria are gaining increasing attention as vaccine platform for their built-in adjuvanticity and for their potential use as carriers of heterologous antigens. These 2 properties offer the opportunity to make highly effective, easy to produce multi-valent vaccines. OMVs can be loaded with foreign antigens by targeting protein expression either to the outer membrane or to the periplasm of the OMV-producing strain. Periplasmic expression is simple and relatively efficient but leads to the accumulation of recombinant antigens in the lumen of OMVs and the ability of OMVs carrying internalized antigens to induce antigen-specific antibody responses has been only marginally investigated and is considered to be sub-optimal.

Methods: We have systematically analyzed in qualitative and quantitative terms antibody responses induced by OMVs carrying different heterologous antigens in their lumen. Group A Streptococcus (GAS) Slo, SpyCEP, Spy0269 and Group B Streptococcus (GBS) SAM_1372 were fused to the OmpA leader sequence for secretion and expressed in Escherichia coli. OMVs from the recombinant strains were purified and tested for immunogenicity and protective activity.

Results: All proteins were incorporated into the OMVs lumen in their native conformation. Upon mice immunization, OMVs induced high functional antibody titers against the recombinant proteins. Furthermore, immunization with Slo-OMVs and SpyCEP-OMVs protected mice against GAS lethal challenge.

Conclusions: The efficiency of antigen delivery to the vesicular lumen via periplasmic expression, and the surprisingly high immunogenicity and protective activity of OMVs carrying internalized recombinant antigens further strengthens the potential of OMVs as vaccine platform.
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http://dx.doi.org/10.3402/jev.v3.24015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4131003PMC
August 2014

SpyAD, a moonlighting protein of group A Streptococcus contributing to bacterial division and host cell adhesion.

Infect Immun 2014 Jul 28;82(7):2890-901. Epub 2014 Apr 28.

Novartis Vaccines and Diagnostics Srl, Siena, Italy.

Group A streptococcus (GAS) is a human pathogen causing a wide repertoire of mild and severe diseases for which no vaccine is yet available. We recently reported the identification of three protein antigens that in combination conferred wide protection against GAS infection in mice. Here we focused our attention on the characterization of one of these three antigens, Spy0269, a highly conserved, surface-exposed, and immunogenic protein of unknown function. Deletion of the spy0269 gene in a GAS M1 isolate resulted in very long bacterial chains, which is indicative of an impaired capacity of the knockout mutant to properly divide. Confocal microscopy and immunoprecipitation experiments demonstrated that the protein was mainly localized at the cell septum and could interact in vitro with the cell division protein FtsZ, leading us to hypothesize that Spy0269 is a member of the GAS divisome machinery. Predicted structural domains and sequence homologies with known streptococcal adhesins suggested that this antigen could also play a role in mediating GAS interaction with host cells. This hypothesis was confirmed by showing that recombinant Spy0269 could bind to mammalian epithelial cells in vitro and that Lactococcus lactis expressing Spy0269 on its cell surface could adhere to mammalian cells in vitro and to mice nasal mucosa in vivo. On the basis of these data, we believe that Spy0269 is involved both in bacterial cell division and in adhesion to host cells and we propose to rename this multifunctional moonlighting protein as SpyAD (Streptococcus pyogenes Adhesion and Division protein).
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http://dx.doi.org/10.1128/IAI.00064-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4097626PMC
July 2014

Protective activity of the CnaBE3 domain conserved among Staphylococcus aureus Sdr proteins.

PLoS One 2013 17;8(9):e74718. Epub 2013 Sep 17.

Novartis Vaccines and Diagnostics, Siena, Italy.

Staphylococcus aureus is an opportunistic pathogen, commensal of the human skin and nares, but also responsible for invasive nosocomial as well as community acquired infections. Staphylococcus aureus adheres to the host tissues by means of surface adhesins, such as SdrC, SdrD, and SdrE proteins. The Sdr family of proteins together with a functional A domain, contain respectively two, three or five repeated sequences called B motifs which comprise the CnaB domains. SdrD and SdrE proteins were reported to be protective in animal models against invasive diseases or lethal challenge with human clinical S. aureus isolates. In this study we identified a 126 amino acid sequence containing a CnaB domain, conserved among the three Sdr proteins. The three fragments defined here as CnaBC2, D5 and E3 domains even though belonging to phylogenetically distinct strains, displayed high sequence similarity. Based on the sequence conservation data, we selected the CnaBE3 domain for further analysis and characterization. Polyclonal antibodies raised against the recombinant CnaBE3 domain recognized SdrE, SdrC and SdrD proteins of different S. aureus lineages. Moreover, we demonstrated that the CnaBE3 domain was expressed in vivo during S. aureus infections, and that immunization of this domain alone significantly reduces the bacterial load in mice challenged with S. aureus. Furthermore, we show that the reduction of bacteria by CnaBE3 vaccination is due to functional antibodies. Finally, we demonstrated that the region of the SdrE protein containing the CnaBE3 domain was resistant to trypsin digestion, a characteristic often associated with the presence of an isopeptide bond.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0074718PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3775735PMC
June 2014

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

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

Novartis Vaccines and Diagnostics, Siena, Italy.

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

Importance: We present a novel example of structural design of a vaccine antigen optimized for human vaccine use. Having previously demonstrated that immunization of mice with streptolysin O elicits a protective immune response against infection with group A Streptococcus strains of different serotypes, we developed in this study a double-mutated nontoxic derivative that represents a novel tool for the development of protective vaccine formulations against this important human pathogen. Furthermore, the innovative construction of an isogenic strain expressing a functionally inactive toxin and its use in infection and opsonophagocytosis experiments allowed us to investigate the mechanism by which streptolysin O mediates protection against group A Streptococcus. Finally, the ability of this toxin to directly attack and kill host immune cells during infection was studied in an air pouch model, which allowed parallel quantification of cellular recruitment, vitality, and cytokine release at the infection site.
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http://dx.doi.org/10.1128/mBio.00387-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3546560PMC
January 2013

Transcriptional regulation of the nadA gene in Neisseria meningitidis impacts the prediction of coverage of a multicomponent meningococcal serogroup B vaccine.

Infect Immun 2013 Feb 10;81(2):560-9. Epub 2012 Dec 10.

Novartis Vaccines and Diagnotics, Research Center, Siena, Italy.

The NadA adhesin is a major component of 4CMenB, a novel vaccine to prevent meningococcus serogroup B (MenB) infection. Under in vitro growth conditions, nadA is repressed by the regulator NadR and poorly expressed, resulting in inefficient killing of MenB strains by anti-NadA antibodies. Interestingly, sera from children infected with strains that express low levels of NadA in laboratory growth nevertheless recognize the NadA antigen, suggesting that NadA expression during infection may be different from that observed in vitro. In a strain panel covering a range of NadA levels, repression was relieved through deleting nadR. All nadR knockout strains expressed high levels of NadA and were efficiently killed by sera from subjects immunized with 4CMenB. A selected MenB strain, NGP165, mismatched for other vaccine antigens, is not killed by sera from immunized infants when the strain is grown in vitro. However, in an in vivo passive protection model, the same sera effectively protected infant rats from bacteremia with NGP165. Furthermore, we identify a novel hydroxyphenylacetic acid (HPA) derivative, reported by others to be produced during inflammation, which induces expression of NadA in vitro, leading to efficient antibody-mediated killing. Finally, using bioluminescent reporters, nadA expression in the infant rat model was induced in vivo at 3 h postinfection. Our results suggest that during infectious disease, NadR repression is alleviated due to niche-specific signals, resulting in high levels of NadA expression from any nadA-positive (nadA(+)) strain and therefore efficient killing by anti-NadA antibodies elicited by the 4CMenB vaccine.
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http://dx.doi.org/10.1128/IAI.01085-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3553804PMC
February 2013

Can we defeat meningococcal disease in low and middle income countries?

Vaccine 2012 May;30 Suppl 2:B63-6

Faculty of Infectious and Tropical Diseases, London School of Hygiene & Tropical Medicine, Keppel St, London WC1E 7HT, UK.

The development of multivalent conjugate and protein-based meningococcal vaccines may make global control of meningococcal disease possible. However, achieving control of meningococcal disease in low and middle income countries will be challenging. In low income countries whose vaccination programmes receive financial support from the Global Alliance for Vaccination and Immunisation, the main challenge is lack of sufficient epidemiological information to allow rational decisions on vaccine introduction to be made and, in these countries, enhanced surveillance is needed. In middle income countries, financial challenges predominate. These could be met by demonstration of the cost effectiveness of new meningococcal vaccines and through the introduction of a tiered-pricing system.
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http://dx.doi.org/10.1016/j.vaccine.2011.12.063DOI Listing
May 2012

Conserved anchorless surface proteins as group A streptococcal vaccine candidates.

J Mol Med (Berl) 2012 Oct 14;90(10):1197-207. Epub 2012 Apr 14.

School of Biological Sciences, University of Wollongong, Wollongong, NSW, 2522, Australia.

Streptococcus pyogenes (group A Streptococcus (GAS)) causes ∼700 million human infections each year, resulting in over 500,000 deaths. The development of a commercial GAS vaccine is hampered by the occurrence of many unique GAS serotypes, antigenic variation within the same serotype, differences in serotype geographical distribution, and the production of antibodies cross-reactive with human tissue that may lead to autoimmune disease. Several independent studies have documented a number of GAS cell wall-associated or secreted metabolic enzymes that contain neither N-terminal leader sequences nor C-terminal cell wall anchors. Here, we applied a proteomic analysis of serotype M1T1 GAS cell wall extracts for the purpose of vaccine development. This approach catalogued several anchorless proteins and identified two protective vaccine candidates, arginine deiminase and trigger factor. These surface-exposed enzymes are expressed across multiple GAS serotypes exhibiting ≥99% amino acid sequence identity. Vaccine safety concerns are alleviated by the observation that these vaccine candidates lack human homologs, while sera from human populations suffering repeated GAS infections and high levels of autoimmune complications do not recognize these enzymes. Our study demonstrates anchorless cell surface antigens as promising vaccine candidates for the prevention of GAS disease.
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http://dx.doi.org/10.1007/s00109-012-0897-9DOI Listing
October 2012

Multi high-throughput approach for highly selective identification of vaccine candidates: the Group A Streptococcus case.

Mol Cell Proteomics 2012 Jun 27;11(6):M111.015693. Epub 2012 Jan 27.

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

We propose an experimental strategy for highly accurate selection of candidates for bacterial vaccines without using in vitro and/or in vivo protection assays. Starting from the observation that efficacious vaccines are constituted by conserved, surface-associated and/or secreted components, the strategy contemplates the parallel application of three high throughput technologies, i.e. mass spectrometry-based proteomics, protein array, and flow-cytometry analysis, to identify this category of proteins, and is based on the assumption that the antigens identified by all three technologies are the protective ones. When we tested this strategy for Group A Streptococcus, we selected a total of 40 proteins, of which only six identified by all three approaches. When the 40 proteins were tested in a mouse model, only six were found to be protective and five of these belonged to the group of antigens in common to the three technologies. Finally, a combination of three protective antigens conferred broad protection against a panel of four different Group A Streptococcus strains. This approach may find general application as an accelerated and highly accurate path to bacterial vaccine discovery.
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http://dx.doi.org/10.1074/mcp.M111.015693DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3433891PMC
June 2012

Surface interactome in Streptococcus pyogenes.

Mol Cell Proteomics 2012 Apr 22;11(4):M111.015206. Epub 2011 Dec 22.

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

Very few studies have so far been dedicated to the systematic analysis of protein interactions occurring between surface and/or secreted proteins in bacteria. Such interactions are expected to play pivotal biological roles that deserve investigation. Taking advantage of the availability of a detailed map of surface and secreted proteins in Streptococcus pyogenes (group A Streptococcus (GAS)), we used protein array technology to define the "surface interactome" in this important human pathogen. Eighty-three proteins were spotted on glass slides in high density format, and each of the spotted proteins was probed for its capacity to interact with any of the immobilized proteins. A total of 146 interactions were identified, 25 of which classified as "reciprocal," namely, interactions that occur irrespective of which of the two partners was immobilized on the chip or in solution. Several of these interactions were validated by surface plasmon resonance and supported by confocal microscopy analysis of whole bacterial cells. By this approach, a number of interesting interactions have been discovered, including those occurring between OppA, DppA, PrsA, and TlpA, proteins known to be involved in protein folding and transport. These proteins, all localizing at the septum, might be part, together with HtrA, of the recently described ExPortal complex of GAS. Furthermore, SpeI was found to strongly interact with the metal transporters AdcA and Lmb. Because SpeI strictly requires zinc to exert its function, this finding provides evidence on how this superantigen, a major player in GAS pathogenesis, can acquire the metal in the host environment, where it is largely sequestered by carrier proteins. We believe that the approach proposed herein can lead to a deeper knowledge of the mechanisms underlying bacterial invasion, colonization, and pathogenesis.
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http://dx.doi.org/10.1074/mcp.M111.015206DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3322576PMC
April 2012

Evaluation of a Group A Streptococcus synthetic oligosaccharide as vaccine candidate.

Vaccine 2010 Dec 24;29(1):104-14. Epub 2010 Sep 24.

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

Bacterial infections caused by Group A Streptococcus (GAS) are a serious health care concern that currently cannot be prevented by vaccination. The GAS cell-wall polysaccharide (GAS-PS) is an attractive vaccine candidate due to its constant expression pattern on different bacterial strains and protective properties of anti-GAS-PS antibodies. Here we report for the first time the immunoprotective efficacy of glycoconjugates with synthetic GAS oligosaccharides as compared to those containing the native GAS-PS. A series of hexa- and dodecasaccharides based on the GAS-PS structure were prepared by chemical synthesis and conjugated to CRM(197). When tested in mice, the conjugates containing the synthetic oligosaccharides conferred levels of immunoprotection comparable to those elicited by the native conjugate. Antisera from immunized rabbits promoted phagocytosis of encapsulated GAS strains. Furthermore we discuss variables that might correlate with glycoconjugate immunogenicity and demonstrate the potential of the synthetic approach that benefits from increased antigen purity and facilitated manufacturing.
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http://dx.doi.org/10.1016/j.vaccine.2010.09.018DOI Listing
December 2010