Publications by authors named "Gianmarco Gasperini"

9 Publications

  • Page 1 of 1

Effect of O-Antigen Chain Length Regulation on the Immunogenicity of and Generalized Modules for Membrane Antigens (GMMA).

Int J Mol Sci 2021 Jan 28;22(3). Epub 2021 Jan 28.

GSK Vaccines Institute for Global Health (GVGH) s.r.l, Via Fiorentina 1, 53100 Siena, Italy.

Recently, generalized modules for membrane antigens (GMMA) technology has been proposed as an alternative approach to traditional glycoconjugate vaccines for O-antigen delivery. Saccharide length is a well-known parameter that can impact the immune response induced by glycoconjugates both in terms of magnitude and quality. However, the criticality of O-antigen length on the immune response induced by GMMA-based vaccines has not been fully elucidated. Here, and GMMA-producing strains were further mutated in order to display homogeneous polysaccharide populations of different sizes on a GMMA surface. Resulting GMMA were compared in mice immunization studies. Athymic nude mice were also used to investigate the involvement of T-cells in the immune response elicited. In contrast with what has been reported for traditional glycoconjugate vaccines and independent of the pathogen and the sugar structural characteristics, O-antigen length did not result in being a critical parameter for GMMA immunogenicity. This work supports the identification of critical quality attributes to optimize GMMA vaccine design and improve vaccine efficacy and gives insights on the nature of the immune response induced by GMMA.
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http://dx.doi.org/10.3390/ijms22031309DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7865430PMC
January 2021

Dissecting the contribution of O-Antigen and proteins to the immunogenicity of Shigella sonnei generalized modules for membrane antigens (GMMA).

Sci Rep 2021 Jan 13;11(1):906. Epub 2021 Jan 13.

GSK Vaccines Institute for Global Health (GVGH), via Fiorentina 1, 53100, Siena, Italy.

GMMA are exosomes released from engineered Gram-negative bacteria resembling the composition of outer membranes. We applied the GMMA technology for the development of an O-Antigen (OAg) based vaccine against Shigella sonnei, the most epidemiologically relevant cause of shigellosis. S. sonnei OAg has been identified as a key antigen for protective immunity, and GMMA are able to induce anti-OAg-specific IgG response in animal models and healthy adults. The contribution of protein-specific antibodies induced upon vaccination with GMMA has never been fully elucidated. Anti-protein antibodies are induced in mice upon immunization with either OAg-negative and OAg-positive GMMA. Here we demonstrated that OAg chains shield the bacteria from anti-protein antibody binding and therefore anti-OAg antibodies were the main drivers of bactericidal activity against OAg-positive bacteria. Interestingly, antibodies that are not targeting the OAg are functional against OAg-negative bacteria. The immunodominant protein antigens were identified by proteomic analysis. Our study confirms a critical role of the OAg on the immune response induced by S. sonnei GMMA. However, little is known about OAg length and density regulation during infection and, therefore, protein exposure. Hence, the presence of protein antigens on S. sonnei GMMA represents an added value for GMMA vaccines compared to other OAg-based formulations.
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http://dx.doi.org/10.1038/s41598-020-80421-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7806729PMC
January 2021

Rational Design of a Glycoconjugate Vaccine against Group A .

Int J Mol Sci 2020 Nov 13;21(22). Epub 2020 Nov 13.

GSK Vaccines Institute for Global Health (GVGH), Via Fiorentina 1, 53100 Siena, Italy.

No commercial vaccine is yet available against Group A (GAS), major cause of pharyngitis and impetigo, with a high frequency of serious sequelae in low- and middle-income countries. Group A Carbohydrate (GAC), conjugated to an appropriate carrier protein, has been proposed as an attractive vaccine candidate. Here, we explored the possibility to use GAS Streptolysin O (SLO), SpyCEP and SpyAD protein antigens with dual role of antigen and carrier, to enhance the efficacy of the final vaccine and reduce its complexity. All protein antigens resulted good carrier for GAC, inducing similar anti-GAC IgG response to the more traditional CRM conjugate in mice. However, conjugation to the polysaccharide had a negative impact on the anti-protein responses, especially in terms of functionality as evaluated by an IL-8 cleavage assay for SpyCEP and a hemolysis assay for SLO. After selecting CRM as carrier, optimal conditions for its conjugation to GAC were identified through a Design of Experiment approach, improving process robustness and yield This work supports the development of a vaccine against GAS and shows how novel statistical tools and recent advancements in the field of conjugation can lead to improved design of glycoconjugate vaccines.
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http://dx.doi.org/10.3390/ijms21228558DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7696035PMC
November 2020

GMMA Is a Versatile Platform to Design Effective Multivalent Combination Vaccines.

Vaccines (Basel) 2020 Sep 17;8(3). Epub 2020 Sep 17.

GSK Vaccines Institute for Global Health (GVGH) S.r.l., 53100 Siena, Italy.

Technology platforms are an important strategy to facilitate the design, development and implementation of vaccines to combat high-burden diseases that are still a threat for human populations, especially in low- and middle-income countries, and to address the increasing number and global distribution of pathogens resistant to antimicrobial drugs. Generalized Modules for Membrane Antigens (GMMA), outer membrane vesicles derived from engineered Gram-negative bacteria, represent an attractive technology to design affordable vaccines. Here, we show that GMMA, decorated with heterologous polysaccharide or protein antigens, leads to a strong and effective antigen-specific humoral immune response in mice. Importantly, GMMA promote enhanced immunogenicity compared to traditional formulations (e.g., recombinant proteins and glycoconjugate vaccines), without negative impact to the anti-GMMA immune response. Our findings support the use of GMMA as a "plug and play" technology for the development of effective combination vaccines targeting different bugs at the same time.
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http://dx.doi.org/10.3390/vaccines8030540DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7564227PMC
September 2020

GMMA and Glycoconjugate Approaches Compared in Mice for the Development of a Vaccine against Serotype 6.

Vaccines (Basel) 2020 Apr 3;8(2). Epub 2020 Apr 3.

GSK Vaccines Institute for Global Health (GVGH) S.r.l., via Fiorentina 1, 53100 Siena, Italy.

infections are one of the top causes of diarrhea throughout the world, with being predominant in developing countries. Currently, no vaccines are widely available and increasing levels of multidrug-resistance make a high priority for vaccine development. The serotype-specific O-antigen moiety of lipopolysaccharide has been recognized as a key target for protective immunity, and many O-antigen based candidate vaccines are in development. Recently, the Generalized Modules for Membrane Antigens (GMMA) technology has been proposed as an alternative approach to traditional glycoconjugate vaccines for O-antigen delivery. Here, these two technologies are compared for a vaccine against serotype 6. Genetic strategies for GMMA production, conjugation approaches for linkage of the O-antigen to CRM carrier protein, and a large panel of analytical methods for full vaccine characterization have been put in place. In a head-to-head immunogenicity study in mice, GMMA induced higher anti-O-antigen IgG than glycoconjugate administered without Alhydrogel. When formulated on Alhydrogel, GMMA and glycoconjugate elicited similar levels of persistent anti-O-antigen IgG with bactericidal activity. Glycoconjugates are a well-established bacterial vaccine approach, but can be costly, particularly when multicomponent preparations are required. With similar immunogenicity and a simpler manufacturing process, GMMA are a promising strategy for the development of a vaccine against .
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http://dx.doi.org/10.3390/vaccines8020160DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7349896PMC
April 2020

Dual role of the colonization factor CD2831 in Clostridium difficile pathogenesis.

Sci Rep 2019 04 3;9(1):5554. Epub 2019 Apr 3.

Glaxo Smith Kline Vaccines, Via Fiorentina 1, 53100, Siena, Italy.

Clostridium difficile is a Gram-positive, anaerobic bacterium and the leading cause of antibiotic-associated diarrhea and pseudomembranous colitis. C. difficile modulates its transition from a motile to a sessile lifestyle through a mechanism of riboswitches regulated by cyclic diguanosine monophosphate (c-di-GMP). Previously described as a sortase substrate positively regulated by c-di-GMP, CD2831 was predicted to be a collagen-binding protein and thus potentially involved in sessility. By overexpressing CD2831 in C. difficile and heterologously expressing it on the surface of Lactococcus lactis, here we further demonstrated that CD2831 is a collagen-binding protein, able to bind to immobilized collagen types I, III and V as well as native collagen produced by human fibroblasts. We also observed that the overexpression of CD2831 raises the ability to form biofilm on abiotic surface in both C. difficile and L. lactis. Notably, we showed that CD2831 binds to the collagen-like domain of the human complement component C1q, suggesting a role in preventing complement cascade activation via the classical pathway. This functional characterization places CD2831 in the Microbial Surface Components Recognizing Adhesive Matrix Molecule (MSCRAMMs) family, a class of virulence factors with a dual role in adhesion to collagen-rich tissues and in host immune evasion by binding to human complement components.
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http://dx.doi.org/10.1038/s41598-019-42000-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6447587PMC
April 2019

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

Physiopathological roles of spontaneously released outer membrane vesicles of Bordetella pertussis.

Future Microbiol 2017 11 5;12:1247-1259. Epub 2017 Oct 5.

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

Aim: Bordetella pertussis has been shown to release outer membrane vesicles (OMV) both in vitro and in vivo but little is known about their biological role during the initial phases of B. pertussis infection of the airways.

Results: We have demonstrated that OMV are released by B. pertussis in a human ciliated-airway cell model and purified vesicles can interact with host cells. Binding and uptake are strictly Bvg-regulated and OMV-associated pertussis toxin contributes to host-cell intoxication. Furthermore, we have shown that OMV act as iron-delivery systems complementing the B. pertussis growth defect in iron-limiting conditions.

Conclusion: We have proved that OMV play different roles in B. pertussis physiopathology and we opened new perspectives to be further investigated.
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http://dx.doi.org/10.2217/fmb-2017-0064DOI Listing
November 2017

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

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

GSK Vaccines, Siena, Italy.

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