Publications by authors named "Renzo Alfini"

10 Publications

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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

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

Glycoconjugate vaccines: current approaches towards faster vaccine design.

Expert Rev Vaccines 2019 09 31;18(9):881-895. Epub 2019 Aug 31.

Research Centre, GSK , Siena , Italy.

: Over the last decades, glycoconjugate vaccines have been proven to be a successful strategy to prevent infectious diseases. Many diseases remain to be controlled, especially in developing countries, and emerging antibiotic-resistant bacteria present an alarming public-health threat. The increasing complexity of future vaccines, and the need to accelerate development processes have triggered the development of faster approaches to glycoconjugate vaccines design. : This review provides an overview of recent progress in glycoconjugation technologies toward faster vaccine design. : Among the different emerging approaches, glycoengineering has the potential to combine glycan assembly and conjugation to carrier systems (such as proteins or outer membrane vesicles) in one step, resulting in a simplified manufacturing process and fewer analytical controls. Chemical and enzymatic strategies, and their automation can facilitate glycoepitope identification for vaccine design. Other approaches, such as the liposomal encapsulation of polysaccharides, potentially enable fast and easy combination of numerous antigens in the same formulation. Additional progress is envisaged in the near future, and some of these systems still need to be further validated in humans. In parallel, new strategies are needed to accelerate the vaccine development process, including the associated clinical trials, up to vaccine release onto the market.
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http://dx.doi.org/10.1080/14760584.2019.1657012DOI Listing
September 2019

Comparative immunogenicity and efficacy of equivalent outer membrane vesicle and glycoconjugate vaccines against nontyphoidal .

Proc Natl Acad Sci U S A 2018 10 27;115(41):10428-10433. Epub 2018 Sep 27.

Jenner Institute, Nuffield Department of Medicine, University of Oxford, Oxford OX3 7DQ, United Kingdom.

Nontyphoidal cause a devastating burden of invasive disease in sub-Saharan Africa with high levels of antimicrobial resistance. Vaccination has potential for a major global health impact, but no licensed vaccine is available. The lack of commercial incentive makes simple, affordable technologies the preferred route for vaccine development. Here we compare equivalent Generalized Modules for Membrane Antigens (GMMA) outer membrane vesicles and O-antigen-CRM glycoconjugates to deliver lipopolysaccharide O-antigen in bivalent Typhimurium and Enteritidis vaccines. strains were chosen and deleted to induce GMMA production. O-antigens were extracted from wild-type bacteria and conjugated to CRM Purified GMMA and glycoconjugates were characterized and tested in mice for immunogenicity and ability to reduce infection. GMMA and glycoconjugate O-antigen had similar structural characteristics, O-acetylation, and glucosylation levels. Immunization with GMMA induced higher anti-O-antigen IgG than glycoconjugate administered without Alhydrogel adjuvant. With Alhydrogel, antibody levels were similar. GMMA induced a diverse antibody isotype profile with greater serum bactericidal activity than glycoconjugate, which induced almost exclusively IgG1. Immunization reduced bacterial colonization of mice subsequently infected with Typhimurium numbers were lower in tissues of mice vaccinated with GMMA compared with glycoconjugate. Enteritidis burden in the tissues was similar in mice immunized with either vaccine. With favorable immunogenicity, low cost, and ability to induce functional antibodies and reduce bacterial burden, GMMA offer a promising strategy for the development of a nontyphoidal vaccine compared with established glycoconjugates. GMMA technology is potentially attractive for development of vaccines against other bacteria of global health significance.
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http://dx.doi.org/10.1073/pnas.1807655115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6187145PMC
October 2018

Immunogenicity of a Bivalent Adjuvanted Glycoconjugate Vaccine against Typhimurium and Enteritidis.

Front Immunol 2017 27;8:168. Epub 2017 Feb 27.

Laboratorio di Microbiologia Molecolare e Biotecnologia (LA.M.M.B.), Dipartimento di Biotecnologie Mediche, Università di Siena , Siena , Italy.

serovars Typhimurium and Enteritidis are the predominant causes of invasive non-typhoidal (iNTS) disease. Considering the co-endemicity of . Typhimurium and . Enteritidis, a bivalent vaccine formulation against both pathogens is necessary for protection against iNTS disease, thus investigation of glycoconjugate combination is required. In the present work, we investigated the immune responses induced by . Typhimurium and . Enteritidis monovalent and bivalent glycoconjugate vaccines adjuvanted with aluminum hydroxide (alum) only or in combination with cytosine-phosphorothioate-guanine oligodeoxynucleotide (CpG). Humoral and cellular, systemic and local, immune responses were characterized in two different mouse strains. All conjugate vaccines elicited high levels of serum IgG against the respective O-antigens (OAg) with bactericidal activity. The bivalent conjugate vaccine induced systemic production of antibodies against both . Typhimurium and . Enteritidis OAg. The presence of alum or alum + CpG adjuvants in vaccine formulations significantly increased the serum antigen-specific antibody production. The alum + CpG bivalent vaccine formulation triggered the highest systemic anti-OAg antibodies and also a significant increase of anti-OAg IgG in intestinal washes and fecal samples, with a positive correlation with serum levels. These data demonstrate the ability of monovalent and bivalent conjugate vaccines against . Typhimurium and . Enteritidis to induce systemic and local immune responses in different mouse strains, and highlight the suitability of a bivalent glycoconjugate formulation, especially when adjuvanted with alum + CpG, as a promising candidate vaccine against iNTS disease.
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http://dx.doi.org/10.3389/fimmu.2017.00168DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5326758PMC
February 2017

Toll-Like Receptor Activation by Generalized Modules for Membrane Antigens from Lipid A Mutants of Salmonella enterica Serovars Typhimurium and Enteritidis.

Clin Vaccine Immunol 2016 Apr 4;23(4):304-14. Epub 2016 Apr 4.

Novartis Vaccines Institute for Global Health, S.r.l., Siena, Italy

Invasive nontyphoidal Salmonella (iNTS) disease is a neglected disease with high mortality in children and HIV-positive individuals in sub-Saharan Africa, caused primarily by Africa-specific strains of Salmonella enterica serovars Typhimurium and Enteritidis. A vaccine using GMMA (generalized modules for membrane antigens) fromS.Typhimurium andS.Enteritidis containing lipid A modifications to reduce potential in vivo reactogenicity is under development. GMMA with penta-acylated lipid A showed the greatest reduction in the level of cytokine release from human peripheral blood monocytes from that for GMMA with wild-type lipid A. Deletion of the lipid A modification genes msbB and pagP was required to achieve pure penta-acylation. Interestingly, ΔmsbBΔ pagP GMMA from S. Enteritidis had a slightly higher stimulatory potential than those from S. Typhimurium, a finding consistent with the higher lipopolysaccharide (LPS) content and Toll-like receptor 2 (TLR2) stimulatory potential of the former. Also, TLR5 ligand flagellin was found in Salmonella GMMA. No relevant contribution to the stimulatory potential of GMMA was detected even when the flagellin protein FliC from S. Typhimurium was added at a concentration as high as 10% of total protein, suggesting that flagellin impurities are not a major factor for GMMA-mediated immune stimulation. Overall, the stimulatory potential of S. Typhimurium and S. Enteritidis ΔmsbB ΔpagP GMMA was close to that of Shigella sonnei GMMA, which are currently in phase I clinical trials.
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http://dx.doi.org/10.1128/CVI.00023-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4820502PMC
April 2016

Relationship between antibody susceptibility and lipopolysaccharide O-antigen characteristics of invasive and gastrointestinal nontyphoidal Salmonellae isolates from Kenya.

PLoS Negl Trop Dis 2015 Mar 4;9(3):e0003573. Epub 2015 Mar 4.

Novartis Vaccines Institute for Global Health (NVGH), Siena, Italy.

Background: Nontyphoidal Salmonellae (NTS) cause a large burden of invasive and gastrointestinal disease among young children in sub-Saharan Africa. No vaccine is currently available. Previous reports indicate the importance of the O-antigen of Salmonella lipopolysaccharide for virulence and resistance to antibody-mediated killing. We hypothesised that isolates with more O-antigen have increased resistance to antibody-mediated killing and are more likely to be invasive than gastrointestinal.

Methodology/principal Findings: We studied 192 NTS isolates (114 Typhimurium, 78 Enteritidis) from blood and stools, mostly from paediatric admissions in Kenya 2000-2011. Isolates were tested for susceptibility to antibody-mediated killing, using whole adult serum. O-antigen structural characteristics, including O-acetylation and glucosylation, were investigated. Overall, isolates were susceptible to antibody-mediated killing, but S. Enteritidis were less susceptible and expressed more O-antigen than Typhimurium (p<0.0001 for both comparisons). For S. Typhimurium, but not Enteritidis, O-antigen expression correlated with reduced sensitivity to killing (r = 0.29, 95% CI = 0.10-0.45, p = 0.002). Both serovars expressed O-antigen populations ranging 21-33 kDa average molecular weight. O-antigen from most Typhimurium were O-acetylated on rhamnose and abequose residues, while Enteritidis O-antigen had low or no O-acetylation. Both Typhimurium and Enteritidis O-antigen were approximately 20%-50% glucosylated. Amount of S. Typhimurium O-antigen and O-antigen glucosylation level were inversely related. There was no clear association between clinical presentation and antibody susceptibility, O-antigen level or other O-antigen features.

Conclusion/significance: Kenyan S. Typhimurium and Enteritidis clinical isolates are susceptible to antibody-mediated killing, with degree of susceptibility varying with level of O-antigen for S. Typhimurium. This supports the development of an antibody-inducing vaccine against NTS for Africa. No clear differences were found in the phenotype of isolates from blood and stool, suggesting that the same isolates can cause invasive disease and gastroenteritis. Genome studies are required to understand whether invasive and gastrointestinal isolates differ at the genotypic level.
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http://dx.doi.org/10.1371/journal.pntd.0003573DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4352093PMC
March 2015

Synthesis of 1,2-dihydroxyindolizidines from 1-(2-pyridyl)-2-propen-1-ol.

J Org Chem 2011 Nov 14;76(22):9536-41. Epub 2011 Oct 14.

Dipartimento di Chimica Ugo Schiff, Università di Firenze, Polo Scientifico e Tecnologico, Sesto Fiorentino, Italy.

1-(2-Pyridyl)-2-propen-1-ol, obtained by vinylation of commercially available picolinaldehyde, resulted a good starting material for the synthesis of the indolizidine skeleton. In particular, a simple process involving bromination, reduction, and nucleophilic substitution (via elimination and addition) allowed an easy conversion of the starting material into (±)-lentiginosine in ~27% overall yield.
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http://dx.doi.org/10.1021/jo201830bDOI Listing
November 2011

Reactivity and synthetic applications of 4,5-dicyanopyridazine: an overview.

Molecules 2010 Mar 12;15(3):1722-45. Epub 2010 Mar 12.

Laboratorio di Progettazione, Sintesi e Studio di Eterocicli Biologicamente Attivi (HeteroBioLab), Dipartimento di Chimica, Università di Firenze, Polo Scientifico e Tecnologico, Via della Lastruccia 3/13, I-50019 Sesto Fiorentino, Italy.

Despite the poor reputation of electron-deficient pyridazines in intermolecular Hetero Diels-Alder (HDA) reactions, 4,5-dicyanopyridazine (DCP) showed a surprising reactivity as a heterocyclic azadiene in inverse electron-demand HDA processes with different dienophiles. The use of alkenes, alkynes and enamines as 2p electron counterparts afforded dicyanocyclohexa-1,3-dienes and substituted phthalonitriles, respectively, while the use of suitable bis-dienophiles provides a general strategy for the one-pot synthesis of polycyclic carbo- and hetero-cage systemsthrough pericyclic three-step homodomino processes. HDA reactions with heterocyclic dienophiles allowed direct benzoannelation: in particular, pyrrole and indole derivatives were converted to dicyano-indoles and -carbazoles. In addition an unprecedented reactivity of DCP as a very reactive heterocyclic electrophile at the C-4 carbon was also evidenced: by changing the experimental conditions, cyanopyrrolyl- and cyanoindolyl-pyridazines were obtained through reactions of pyrrole and indole systems as carbon nucleophiles in formal SNAr2 processes where a CN group of DCP acts as leaving group. Thus, careful control of the reaction conditions allows exploitation of both pathways for the synthesis of different classes of heterocyclic derivatives.
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http://dx.doi.org/10.3390/molecules15031722DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6257364PMC
March 2010