Publications by authors named "Giuliano Bensi"

27 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

Outer Membrane Vesicles (OMV)-based and Proteomics-driven Antigen Selection Identifies Novel Factors Contributing to Adhesion to Epithelial Cells.

Mol Cell Proteomics 2018 02 4;17(2):205-215. Epub 2017 Dec 4.

From the ‡GSK Vaccines, Siena, Italy

Despite high vaccination coverage world-wide, whooping cough, a highly contagious disease caused by is recently increasing in occurrence suggesting that novel vaccine formulations targeted at the prevention of colonization and transmission should be investigated. To identify new candidates for inclusion in the acellular formulation, we used spontaneously released outer membrane vesicles (OMV) as a potential source of key adhesins. The enrichment of Bvg+ OMV with adhesins and the ability of anti-OMV serum to inhibit the adhesion of to lung epithelial cells were demonstrated. We employed a proteomic approach to identify the differentially expressed proteins in OMV purified from bacteria in the Bvg+ and Bvg- virulence phases, thus comparing the outer membrane protein pattern of this pathogen in its virulent or avirulent state. Six of the most abundant outer membrane proteins were selected as candidates to be evaluated for their adhesive properties and vaccine potential. We generated strains singularly expressing the selected proteins and assessed their ability to adhere to lung epithelial cells Four out of the selected proteins conferred adhesive ability to Three of the candidates were specifically detected by anti-OMV mouse serum suggesting that these proteins are immunogenic antigens able to elicit an antibody response when displayed on the OMV. Anti-OMV serum was able to inhibit only BrkA-expressing adhesion to lung epithelial cells. Finally, stand-alone immunization of mice with recombinant BrkA resulted in significant protection against infection of the lower respiratory tract after challenge with Taken together, these data support the inclusion of BrkA and possibly further adhesins to the current acellular pertussis vaccines to improve the impact of vaccination on the bacterial clearance.
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http://dx.doi.org/10.1074/mcp.RA117.000045DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5795387PMC
February 2018

Development of a Click Beetle Luciferase Reporter System for Enhanced Bioluminescence Imaging of : Analysis in Cell Culture and Murine Infection Models.

Front Microbiol 2017 26;8:1797. Epub 2017 Sep 26.

APC Microbiome Institute, University College Cork, Cork, Ireland.

is a Gram-positive facultative intracellular pathogen that is widely used as a model organism for the analysis of infection biology. In this context, there is a current need to develop improved reporters for enhanced bioluminescence imaging (BLI) of the pathogen in infection models. We have developed a click beetle red luciferase (CBR-) based vector (pPL2CBR) expressing codon optimized CBR- under the control of a highly expressed Listerial promoter (P) for and have compared this to a -based system expressing bacterial luciferase for BLI of the pathogen using growth experiments and models. The CBR- plasmid stably integrates into the chromosome and can be used to label field isolates and laboratory strains of the pathogen. Growth experiments revealed that CBR- labeled emits a bright signal in exponential phase that is maintained during stationary phase. In contrast, -labeled bacteria produced a light signal that peaked during exponential phase and was significantly reduced during stationary phase. Light from CBR- labeled bacteria was more efficient than the signal from -labeled bacteria in penetrating an artificial tissue depth assay system. A cell invasion assay using C2Bbe1 cells and a systemic murine infection model revealed that CBR- is suited to BLI approaches and demonstrated enhanced sensitivity relative to in the context of infection models. Overall, we demonstrate that this novel CBR reporter system provides efficient, red-shifted light production relative to and may have significant applications in the analysis of pathogenesis.
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http://dx.doi.org/10.3389/fmicb.2017.01797DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5622934PMC
September 2017

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

One Dose of Staphylococcus aureus 4C-Staph Vaccine Formulated with a Novel TLR7-Dependent Adjuvant Rapidly Protects Mice through Antibodies, Effector CD4+ T Cells, and IL-17A.

PLoS One 2016 26;11(1):e0147767. Epub 2016 Jan 26.

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

A rapidly acting, single dose vaccine against Staphylococcus aureus would be highly beneficial for patients scheduled for major surgeries or in intensive care units. Here we show that one immunization with a multicomponent S. aureus candidate vaccine, 4C-Staph, formulated with a novel TLR7-dependent adjuvant, T7-alum, readily protected mice from death and from bacterial dissemination, both in kidney abscess and peritonitis models, outperforming alum-formulated vaccine. This increased efficacy was paralleled by higher vaccine-specific and α-hemolysin-neutralizing antibody titers and Th1/Th17 cell responses. Antibodies played a crucial protective role, as shown by the lack of protection of 4C-Staph/T7-alum vaccine in B-cell-deficient mice and by serum transfer experiments. Depletion of effector CD4+ T cells not only reduced survival but also increased S. aureus load in kidneys of mice immunized with 4C-Staph/T7-alum. The role of IL-17A in the control of bacterial dissemination in 4C-Staph/T7-alum vaccinated mice was indicated by in vivo neutralization experiments. We conclude that single dose 4C-Staph/T7-alum vaccine promptly and efficiently protected mice against S. aureus through the combined actions of antibodies, CD4+ effector T cells, and IL-17A. These data suggest that inclusion of an adjuvant that induces not only fast antibody responses but also IL-17-producing cell-mediated effector responses could efficaciously protect patients scheduled for major surgeries or in intensive care units.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0147767PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4727907PMC
July 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

Four-component Staphylococcus aureus vaccine 4C-staph enhances Fcγ receptor expression in neutrophils and monocytes and mitigates S. aureus infection in neutropenic mice.

Infect Immun 2015 Aug 26;83(8):3157-63. Epub 2015 May 26.

Novartis Vaccines and Diagnostics, s.r.l., a GSK Company, Siena, Italy

Staphylococcus aureus is a human bacterial pathogen causing a variety of diseases. The occurrence of multidrug-resistant strains of Staphylococcus aureus underlines the need for a vaccine. Defining immune correlates of protection may support the design of an effective vaccine. We used a murine Staphylococcus aureus infection model, in which bacteria were inoculated in an air pouch generated on the back of the animal. Analysis of the air-pouch content in mice immunized or not with an adjuvanted multiantigen vaccine formulation, four-component S. aureus vaccine (4C-Staph), prior to infection allowed us to measure bacteria, cytokines, and 4C-Staph-specific antibodies and to analyze host immune cells recruited to the infection site. Immunization with 4C-Staph resulted in accumulation of antigen-specific antibodies in the pouch and mitigated the infection. Neutrophils were the most abundant cells in the pouch, and they showed the upregulation of Fcγ receptor (FcγR) following immunization with 4C-Staph. Reduction of the infection was also obtained in mice immunized with 4C-Staph and depleted of neutrophils; these mice showed an increase in monocytes and macrophages. Upregulation of the FcγR and the presence of antigen-specific antibodies induced by immunization with 4C-Staph may contribute to increase bacterial opsonophagocytosis. Protection in neutropenic mice indicated that an effective vaccine could activate alternative protection mechanisms compensating for neutropenia, a condition often occurring in S. aureus-infected patients.
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http://dx.doi.org/10.1128/IAI.00258-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4496606PMC
August 2015

Vaccine composition formulated with a novel TLR7-dependent adjuvant induces high and broad protection against Staphylococcus aureus.

Proc Natl Acad Sci U S A 2015 Mar 9;112(12):3680-5. Epub 2015 Mar 9.

Novartis Vaccines Research Center, 53100 Siena, Italy;

Both active and passive immunization strategies against Staphylococcus aureus have thus far failed to show efficacy in humans. With the attempt to develop an effective S. aureus vaccine, we selected five conserved antigens known to have different roles in S. aureus pathogenesis. They include the secreted factors α-hemolysin (Hla), ess extracellular A (EsxA), and ess extracellular B (EsxB) and the two surface proteins ferric hydroxamate uptake D2 and conserved staphylococcal antigen 1A. The combined vaccine antigens formulated with aluminum hydroxide induced antibodies with opsonophagocytic and functional activities and provided consistent protection in four mouse models when challenged with a panel of epidemiologically relevant S. aureus strains. The importance of antibodies in protection was demonstrated by passive transfer experiments. Furthermore, when formulated with a toll-like receptor 7-dependent (TLR7) agonist recently designed and developed in our laboratories (SMIP.7-10) adsorbed to alum, the five antigens provided close to 100% protection against four different staphylococcal strains. The new formulation induced not only high antibody titers but also a Th1 skewed immune response as judged by antibody isotype and cytokine profiles. In addition, low frequencies of IL-17-secreting T cells were also observed. Altogether, our data demonstrate that the rational selection of mixtures of conserved antigens combined with Th1/Th17 adjuvants can lead to promising vaccine formulations against S. aureus.
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http://dx.doi.org/10.1073/pnas.1424924112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4378396PMC
March 2015

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

SslE elicits functional antibodies that impair in vitro mucinase activity and in vivo colonization by both intestinal and extraintestinal Escherichia coli strains.

PLoS Pathog 2014 May 8;10(5):e1004124. Epub 2014 May 8.

Novartis Vaccines and Diagnostics Srl, Siena, Italy.

SslE, the Secreted and surface-associated lipoprotein from Escherichia coli, has recently been associated to the M60-like extracellular zinc-metalloprotease sub-family which is implicated in glycan recognition and processing. SslE can be divided into two main variants and we recently proposed it as a potential vaccine candidate. By applying a number of in vitro bioassays and comparing wild type, knockout mutant and complemented strains, we have now demonstrated that SslE specifically contributes to degradation of mucin substrates, typically present in the intestine and bladder. Mutation of the zinc metallopeptidase motif of SslE dramatically impaired E. coli mucinase activity, confirming the specificity of the phenotype observed. Moreover, antibodies raised against variant I SslE, cloned from strain IHE3034 (SslEIHE3034), are able to inhibit translocation of E. coli strains expressing different variants through a mucin-based matrix, suggesting that SslE induces cross-reactive functional antibodies that affect the metallopeptidase activity. To test this hypothesis, we used well-established animal models and demonstrated that immunization with SslEIHE3034 significantly reduced gut, kidney and spleen colonization by strains producing variant II SslE and belonging to different pathotypes. Taken together, these data strongly support the importance of SslE in E. coli colonization of mucosal surfaces and reinforce the use of this antigen as a component of a broadly protective vaccine against pathogenic E. coli species.
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http://dx.doi.org/10.1371/journal.ppat.1004124DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4014459PMC
May 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

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

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

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

Protein array profiling of tic patient sera reveals a broad range and enhanced immune response against Group A Streptococcus antigens.

PLoS One 2009 Jul 22;4(7):e6332. Epub 2009 Jul 22.

Novartis Vaccines and Diagnostics, Siena, Italy.

The human pathogen Group A Streptococcus (Streptococcus pyogenes, GAS) is widely recognized as a major cause of common pharyngitis as well as of severe invasive diseases and non-suppurative sequelae associated with the existence of GAS antigens eliciting host autoantibodies. It has been proposed that a subset of paediatric disorders characterized by tics and obsessive-compulsive symptoms would exacerbate in association with relapses of GAS-associated pharyngitis. This hypothesis is however still controversial. In the attempt to shed light on the contribution of GAS infections to the onset of neuropsychiatric or behavioral disorders affecting as many as 3% of children and adolescents, we tested the antibody response of tic patient sera to a representative panel of GAS antigens. In particular, 102 recombinant proteins were spotted on nitrocellulose-coated glass slides and probed against 61 sera collected from young patients with typical tic neuropsychiatric symptoms but with no overt GAS infection. Sera from 35 children with neither tic disorder nor overt GAS infection were also analyzed. The protein recognition patterns of these two sera groups were compared with those obtained using 239 sera from children with GAS-associated pharyngitis. This comparative analysis identified 25 antigens recognized by sera of the three patient groups and 21 antigens recognized by tic and pharyngitis sera, but poorly or not recognized by sera from children without tic. Interestingly, these antigens appeared to be, in quantitative terms, more immunogenic in tic than in pharyngitis patients. Additionally, a third group of antigens appeared to be preferentially and specifically recognized by tic sera. These findings provide the first evidence that tic patient sera exhibit immunological profiles typical of individuals who elicited a broad, specific and strong immune response against GAS. This may be relevant in the context of one of the hypothesis proposing that GAS antigen-dependent induction of autoantibodies in susceptible individuals may be involved the occurrence of tic disorders.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0006332PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2709431PMC
July 2009

Scavenger receptor gp340 aggregates group A streptococci by binding pili.

Mol Microbiol 2008 Jun 29;68(6):1378-94. Epub 2008 Apr 29.

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

Group A streptococci (GAS) are the most frequent cause of bacterial pharyngitis. The first obstacle to GAS colonization of the pharynx is saliva. As well as forming a physical barrier, saliva contains components of innate and acquired immunity. Previous work has shown that saliva induces bacterial aggregation, which may serve as a clearance mechanism. As the aggregation of some oral streptococci in saliva is mediated by long proteinaceous appendages, we hypothesized that pili of GAS might behave similarly. Wild-type GAS M1 strain SF370 aggregated in saliva, while pilus-defective mutants did not. Similarly, heterologous expression of diverse GAS pili on the surface of Lactococcus lactis induced aggregation in saliva, while control strains were unaffected. Further studies revealed that aggregating bacteria bound salivary component gp340. Purified gp340 aggregated wild-type GAS and L. lactis expressing GAS pili, but not control strains. GAS pilus-defective mutants were abrogated in gp340 binding and aggregation. Furthermore, gp340-mediated aggregation reduced bacterial adhesion to human epithelial cells, suggesting a role in host defence.
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http://dx.doi.org/10.1111/j.1365-2958.2008.06220.xDOI Listing
June 2008

Streptococcus pyogenes pili promote pharyngeal cell adhesion and biofilm formation.

Mol Microbiol 2007 May;64(4):968-83

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

Group A Streptococcus (GAS, Streptococcus pyogenes) is a Gram-positive human pathogen responsible for several acute diseases and autoimmune sequelae that account for half a million deaths worldwide every year. GAS infections require the capacity of the pathogen to adhere to host tissues and assemble in cell aggregates. Furthermore, a role for biofilms in GAS pathogenesis has recently been proposed. Here we investigated the role of GAS pili in biofilm formation. We demonstrated that GAS pilus-negative mutants, in which the genes encoding either the pilus backbone structural protein or the sortase C1 have been deleted, showed an impaired capacity to attach to a pharyngeal cell line. The same mutants were much less efficient in forming cellular aggregates in liquid culture and microcolonies on human cells. Furthermore, mutant strains were incapable of producing the typical three-dimensional layer with bacterial microcolonies embedded in a carbohydrate polymeric matrix. Complemented mutants had an adhesion and aggregation phenotype similar to the wild-type strain. Finally, in vivo expression of pili was indirectly confirmed by demonstrating that most of the sera from human patients affected by GAS-mediated pharyngitis recognized recombinant pili proteins. These data support the role of pili in GAS adherence and colonization and suggest a general role of pili in all pathogenic streptococci.
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http://dx.doi.org/10.1111/j.1365-2958.2007.05704.xDOI Listing
May 2007

Characterization and identification of vaccine candidate proteins through analysis of the group A Streptococcus surface proteome.

Nat Biotechnol 2006 Feb 15;24(2):191-7. Epub 2006 Jan 15.

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

We describe a proteomic approach for identifying bacterial surface-exposed proteins quickly and reliably for their use as vaccine candidates. Whole cells are treated with proteases to selectively digest protruding proteins that are subsequently identified by mass spectrometry analysis of the released peptides. When applied to the sequenced M1_SF370 group A Streptococcus strain, 68 PSORT-predicted surface-associated proteins were identified, including most of the protective antigens described in the literature. The number of surface-exposed proteins varied from strain to strain, most likely as a consequence of different capsule content. The surface-exposed proteins of the highly virulent M23_DSM2071 strain included 17 proteins, 15 in common with M1_SF370. When 14 of the 17 proteins were expressed in E. coli and tested in the mouse for their capacity to confer protection against a lethal dose of M23_DSM2071, one new protective antigen (Spy0416) was identified. This strategy overcomes the difficulties so far encountered in surface protein characterization and has great potential in vaccine discovery.
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http://dx.doi.org/10.1038/nbt1179DOI Listing
February 2006

Group A Streptococcus produce pilus-like structures containing protective antigens and Lancefield T antigens.

Proc Natl Acad Sci U S A 2005 Oct 13;102(43):15641-6. Epub 2005 Oct 13.

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

Although pili have long been recognized in Gram-negative pathogens as important virulence factors involved in adhesion and invasion, very little is known about extended surface organelles in Gram-positive pathogens. Here we report that Group A Streptococcus (GAS), a Gram-positive human-specific pathogen that causes pharyngitis, impetigo, invasive disease, necrotizing fasciitis, and autoimmune sequelae has long, surface-exposed, pilus-like structures composed of members of a family of extracellular matrix-binding proteins. We describe four variant pili and show that each is recognized by a specific serum of the Lancefield T-typing system, which has been used for over five decades to characterize GAS isolates. Furthermore, we show that immunization of mice with a combination of recombinant pilus proteins confers protection against mucosal challenge with virulent GAS bacteria. The data indicate that induction of a protective immune response against these structures may be a useful strategy for development of a vaccine against disease caused by GAS infection.
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http://dx.doi.org/10.1073/pnas.0507808102DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1253647PMC
October 2005

Chlamydia pneumoniae genome sequence analysis and identification of HLA-A2-restricted CD8+ T cell epitopes recognized by infection-primed T cells.

Vaccine 2005 Oct;23(42):5028-37

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

In the present study, we performed in silico analysis of Chlamydia pneumoniae genome sequence to identify human HLA-A2-restricted T cell epitopes. Thirty-one Chlamydia-specific protein antigens were selected and peptides were derived thereof using an HLA-A2 epitope predictive algorithm. Firstly, we tested binding of 55 selected 9mer peptides to HLA-A2 in vitro. Next, infection of HLA-A2 transgenic mice with C. pneumoniae elementary bodies and assessment of effector CD8+ T cells allowed us to identify which of the epitopes binding to HLA-A2 in vitro were recognized by C. pneumoniae infection-primed CD8+ T cells. Finally, we could confirm that CD8+ T cells in association with HLA-A2 recognized the most reactive peptides when the corresponding full-length genes were used to DNA-immunize HLA-A2 transgenic mice. By using this approach, a novel HLA-A2-restricted epitope in the outer membrane protein A (OmpA) of C. pneumoniae was identified, which proved to mediate specific lysis of peptide-loaded target cells.
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http://dx.doi.org/10.1016/j.vaccine.2005.04.048DOI Listing
October 2005

Effect of twelve-months therapy with oral ambroxol in preventing exacerbations in patients with COPD. Double-blind, randomized, multicenter, placebo-controlled study (the AMETHIST Trial).

Pulm Pharmacol Ther 2004 ;17(1):27-34

Dipartimento di Medicina Interna, 1 Divisione di Medicina, University of Brescia, Spedali Civili, p.le Spedali Civili, 25100 Brescia, Italy.

The objective of this prospective, randomized, double-blind, placebo-controlled, multicenter parallel-group study was to evaluate the effect of long-term ambroxol treatment in preventing exacerbations of chronic obstructive pulmonary disease (COPD). Two hundred and forty-two outpatients with COPD defined by ATS criteria with value of FEV1 between > or =60 and 80% of predicted and history of one or more exacerbations in the previous year were recruited by 26 Respiratory Medicine Centers in Italy and treated for 1 year with one ambroxol retard capsule of 75 mg twice daily or placebo. The percentage of patients free from exacerbation at 6 months was 63% with ambroxol and 60% with placebo (p=0.366) and at 12 months 56% with ambroxol and 53% with placebo (p=0.363). In a subset of 45 patients with more severe baseline symptoms, ambroxol therapy was associated with a significant higher percentage of patients free from exacerbation compared to placebo: 63 vs. 38% (p=0.038). In conclusion, we did not find a significant difference between long-term ambroxol therapy and placebo, in preventing exacerbations in patients with COPD. In patients with more severe respiratory symptoms at baseline, however, we observed a significant difference in the cumulative exacerbation-free persistence between ambroxol and placebo, suggesting that long-term muco-regulatory therapy with ambroxol could be useful in highly symptomatic patients with COPD.
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http://dx.doi.org/10.1016/j.pupt.2003.08.004DOI Listing
April 2004

Yeast coexpression of human papillomavirus types 6 and 16 capsid proteins.

Virology 2002 Feb;293(2):335-44

Department of Molecular Biology, IRIS, Chiron S.p.A., Via Fiorentina 1, 53100 Siena, Italy.

The L1 and L2 capsid proteins of animal and human papillomaviruses (HPVs) can self-assemble into virus-like particles (VLPs) that closely resemble native virions. The use of different animal models shows that VLPs can be very efficient at inducing a protective immune response. However, studies with infectious HPV virions and VLPs of different HPV types indicate that the immune response is predominantly type-specific. We have generated a diploid yeast strain that coexpresses the L1 and L2 capsid proteins of both HPV-6b and HPV-16, and we have purified fully assembled VLPs banding in a cesium chloride gradient at the expected density of 1.29-1.3 mg/ml. Experimental evidence strongly indicated that the four proteins coassembled into VLPs. Western blot analysis, using anti-HPV-6 and anti-HPV-16 L1-specific monoclonal antibodies and type-specific L2 antisera, demonstrated that all four proteins copurified. Most importantly, immunoprecipitation experiments, carried out using type-specific anti-L1 monoclonals and either total yeast cell extracts or purified VLPs, confirmed the interaction and the formation of covalent disulfide bonds between the two L1 proteins. Finally, HPV-6/16 VLPs administered to mice induced conformational antibodies against both L1 protein types. These results suggest that coexpression of different capsid proteins may provide new tools for the induction of antibodies directed against multiple HPV types.
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http://dx.doi.org/10.1006/viro.2001.1289DOI Listing
February 2002