Publications by authors named "Anna Maria Colucci"

8 Publications

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The role of vaccines and vaccine decision-making to achieve the goals of the Grand Convergence in public health.

Vaccine 2017 01 22;35 Suppl 1:A10-A15. Epub 2016 Dec 22.

Imperial College, London, England. Electronic address:

On 17 and 18 July 2015, a meeting in Siena jointly sponsored by ADITEC and GlaxoSmithKline (GSK) was held to review the goals of the Global Health 2035 Grand Convergence, to discuss current vaccine evaluation methods, and to determine the feasibility of reaching consensus on an assessment framework for comprehensively and accurately capturing the full benefits of vaccines. Through lectures and workshops, participants reached a consensus that Multi-Criteria-Decision-Analysis is a method suited to systematically account for the many variables needed to evaluate the broad benefits of vaccination, which include not only health system savings, but also societal benefits, including benefits to the family and increased productivity. Participants also agreed on a set of "core values" to be used in future assessments of vaccines for development and introduction. These values include measures of vaccine efficacy and safety, incident cases prevented per year, the results of cost-benefit analyses, preventable mortality, and the severity of the target disease. Agreement on this set of core assessment parameters has the potential to increase alignment between manufacturers, public health agencies, non-governmental organizations (NGOs), and policy makers (see Global Health 2035 Mission Grand Convergence [1]). The following sections capture the deliberations of a workshop (Working Group 4) chartered to: (1) review the list of 24 parameters selected from SMART vaccines (see the companion papers by Timmis et al. and Madhavan et al., respectively) to determine which represent factors (see Table 1) that should be taken into account when evaluating the role of vaccines in maximizing the success of the Global Health 2035 Grand Convergence; (2) develop 3-5 "core values" that should be taken into account when evaluating vaccines at various stages of development; and (3) determine how vaccines can best contribute to the Global Health 2035 Grand Convergence effort.
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http://dx.doi.org/10.1016/j.vaccine.2016.10.088DOI Listing
January 2017

Production of a Shigella sonnei Vaccine Based on Generalized Modules for Membrane Antigens (GMMA), 1790GAHB.

PLoS One 2015 6;10(8):e0134478. Epub 2015 Aug 6.

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

Recently, we developed a high yield production process for outer membrane particles from genetically modified bacteria, called Generalized Modules of Membrane Antigens (GMMA), and the corresponding simple two step filtration purification, enabling economic manufacture of these particles for use as vaccines. Using a Shigella sonnei strain that was genetically modified to produce penta-acylated lipopolysaccharide (LPS) with reduced endotoxicity and to maintain the virulence plasmid encoding for the immunodominant O antigen component of the LPS, scale up of the process to GMP pilot scale was straightforward and gave high yields of GMMA with required purity and consistent results. GMMA were formulated with Alhydrogel and were highly immunogenic in mice and rabbits. In mice, a single immunization containing 29 ng protein and 1.75 ng of O antigen elicited substantial anti-LPS antibody levels. As GMMA contain LPS and lipoproteins, assessing potential reactogenicity was a key aspect of vaccine development. In an in vitro monocyte activation test, GMMA from the production strain showed a 600-fold lower stimulatory activity than GMMA with unmodified LPS. Two in vivo tests confirmed the low potential for reactogenicity. We established a modified rabbit pyrogenicity test based on the European Pharmacopoeia pyrogens method but using intramuscular administration of the full human dose (100 μg of protein). The vaccine elicited an average temperature rise of 0.5°C within four hours after administration, which was considered acceptable and showed that the test is able to detect a pyrogenic response. Furthermore, a repeat dose toxicology study in rabbits using intramuscular (100 μg/dose), intranasal (80 μg/dose), and intradermal (10 μg/dose) administration routes showed good tolerability of the vaccine by all routes and supported its suitability for use in humans. The S. sonnei GMMA vaccine is now in Phase 1 dose-escalation clinical trials.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0134478PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4527750PMC
May 2016

Simplified low-cost production of O-antigen from Salmonella Typhimurium Generalized Modules for Membrane Antigens (GMMA).

J Biotechnol 2015 Mar 3;198:46-52. Epub 2015 Feb 3.

Novartis Vaccines Institute for Global Health, Via Fiorentina 1, 53100 Siena, Italy. Electronic address:

The Novartis Vaccines Institute for Global Health is developing vaccines using outer membrane particles, known as Generalized Modules for Membrane Antigens (GMMA). These are blebs of outer membrane and periplasm, shed from the surface of living Gram-negative bacteria following the targeted deletion of proteins involved in maintaining the integrity of the inner and outer membranes. The current study investigates the use of GMMA as starting material for extraction of membrane components, focusing on the O-antigen polysaccharide portion of lipopolysaccharide from Salmonella Typhimurium. We show that the amount of O-antigen extracted from GMMA by acid hydrolysis is comparable to the quantity extracted from whole wild type bacteria, but with less protein and DNA contaminants. Compared to conventional purification, GMMA enabled a reduction in the number of purification steps required to obtain the O-antigen polysaccharide with the same purity. Purification processes from GMMA and bacteria were characterised by similar final yields. Use of GMMA as starting material provides the possibility to simplify the purification process of O-antigen, with a consequent decrease in manufacturing costs of O-antigen-based glyconjugate vaccines against Salmonella strains and potentially other Gram-negative bacteria.
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http://dx.doi.org/10.1016/j.jbiotec.2015.01.020DOI Listing
March 2015

High yield production process for Shigella outer membrane particles.

PLoS One 2012 6;7(6):e35616. Epub 2012 Jun 6.

Novartis Vaccines Institute for Global Health, Siena, Italy.

Gram-negative bacteria naturally shed particles that consist of outer membrane lipids, outer membrane proteins, and soluble periplasmic components. These particles have been proposed for use as vaccines but the yield has been problematic. We developed a high yielding production process of genetically derived outer membrane particles from the human pathogen Shigella sonnei. Yields of approximately 100 milligrams of membrane-associated proteins per liter of fermentation were obtained from cultures of S. sonnei ΔtolR ΔgalU at optical densities of 30-45 in a 5 L fermenter. Proteomic analysis of the purified particles showed the preparation to primarily contain predicted outer membrane and periplasmic proteins. These were highly immunogenic in mice. The production of these outer membrane particles from high density cultivation of bacteria supports the feasibility of scaling up this approach as an affordable manufacturing process. Furthermore, we demonstrate the feasibility of using this process with other genetic manipulations e.g. abolition of O antigen synthesis and modification of the lipopolysaccharide structure in order to modify the immunogenicity or reactogenicity of the particles. This work provides the basis for a large scale manufacturing process of Generalized Modules of Membrane Antigens (GMMA) for production of vaccines from gram-negative bacteria.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0035616PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3368891PMC
November 2012

Dictyostelium discoideum as a model host for meningococcal pathogenesis.

Med Sci Monit 2008 Jul;14(7):BR134-40

Department of Biological and Environmental Sciences and Technologies, DiSTeBA, University of Salento, Lecce, Italy.

Background: The aim of the present study was to evaluate the possibility of studying meningococcal virulence in a new model organism, Dictyostelium discoideum, a haploid social soil amoeba that is an established host model for several human pathogens, leading to the discovery of novel virulence mechanisms.

Material/methods: A number of virulent and hyper-virulent N. meningitidis strains, including isogenic encapsulated, unencapsulated, and lipooligosaccharide (LOS) outer core-defective derivatives, were used to test the ability of D. discoideum to internalize and grow in the presence of bacteria. Intracellular survival of the internalized bacteria was also monitored.

Results: Meningococci were internalized and killed by D. discoideum cells. The presence of a capsule did not affect the internalization, but, as in human cells, it increased the resistance of the internalized bacteria. Although both encapsulated and unencapsulated meningococci supported the growth and development of D. discoideum on an agar surface, in liquid medium the encapsulated strains were toxic to the slime mould cells. Toxicity inversely correlated with meningococcal survival in the assay medium that was not favorable to bacterial replication, suggesting that it may be due to some toxic compound released after bacterial autolysis. Intriguingly, unencapsulated isogenic strains efficiently supported Dictyostelium growth in suspension, opening the possibility that the toxicity may be associated with the capsular polysaccharide.

Conclusions: These results suggest that several meningococcal virulence determinants, such as the capsular polysaccharide, may be remarkably effective also in Dictyostelium cells, stimulating the use of this model host to search for novel meningococcal virulence determinants.
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July 2008

Functional characterization of Rab7 mutant proteins associated with Charcot-Marie-Tooth type 2B disease.

J Neurosci 2008 Feb;28(7):1640-8

Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali, Università del Salento, 73100 Lecce, Italy.

Charcot-Marie-Tooth (CMT) type 2 neuropathies are a group of autosomal-dominant axonal disorders genetically and clinically heterogeneous. In particular, CMT type 2B (CMT2B) neuropathies are characterized by severe sensory loss, often complicated by infections, arthropathy, and amputations. Recently, four missense mutations in the small GTPase Rab7 associated with the Charcot-Marie Tooth type 2B phenotype have been identified. These mutations target highly conserved amino acid residues. However, nothing is known about whether and how these mutations affect Rab7 function. We investigated the biochemical and functional properties of three of the mutant proteins. Interestingly, all three proteins exhibited higher nucleotide exchange rates and hydrolyzed GTP slower than the wild-type protein. In addition, whereas 23% of overexpressed wild-type Rab7 was GTP bound in HeLa cells, the large majority of the mutant proteins (82-89%) were in the GTP-bound form, consistent with the data on GTP hydrolysis and exchange rates. The CMT2B-associated Rab7 proteins were also able to bind the Rab7 effector RILP (Rab-interacting lysosomal protein) and to rescue Rab7 function after silencing. Altogether, these data demonstrate that all tested CMT2B-associated Rab7 mutations are mechanistically similar, suggesting that activated forms of the Rab7 are responsible for CMT2B disease.
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http://dx.doi.org/10.1523/JNEUROSCI.3677-07.2008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6671532PMC
February 2008

Expression, assay, and functional properties of RILP.

Methods Enzymol 2005 ;403:664-75

Rab proteins are master regulators of vesicular membrane traffic of endocytic and exocytic pathways. They basically serve to recruit proteins and lipids required for vesicle formation, docking, and fusion. Each Rab protein is able to recruit one or more effectors, and, through the action of effectors, it drives its specific downstream functions. The Rab interacting lysosomal protein (RILP) is a common effector of Rab7 and Rab34, two Rab proteins implicated in the biogenesis of lysosomes. RILP is recruited onto late endosomal/lysosomal membranes by Rab7-GTP where it induces the recruitment of the dynein-dynactin motor complexes. Therefore, through the timed and selective dynein motor recruitment onto late endosomes and lysosomes, Rab7 and RILP control transport to endocytic degradative compartments. A similar role for Rab7 and RILP has been demonstrated also for phagosomes. Indeed, RILP recruits dynein-dynactin motors on Rab7-GTP-positive phagosomes and the recruitment not only displaces phagosomes centripetally, but also promotes the extension of phagosomal tubules toward late endocytic compartments. RILP is therefore a key protein for the biogenesis of lysosomes and phagolysosomes. This chapter describes how to express wild-type or mutated RILP in mammalian cells, and how to test the effects caused by RILP dysfunction. In particular, we report assays to monitor the interaction between RILP and Rab7, morphology and distribution of endosomes, and to measure degradation of endocytic markers.
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http://dx.doi.org/10.1016/S0076-6879(05)03057-0DOI Listing
April 2006

The Rab-interacting lysosomal protein, a Rab7 and Rab34 effector, is capable of self-interaction.

Biochem Biophys Res Commun 2005 Aug;334(1):128-33

Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali, Università degli Studi di Lecce, Via Monteroni, 73100 Lecce, Italy.

Rab-interacting lysosomal protein (RILP) has been identified as an interacting partner of the small GTPases Rab7 and Rab34. Active Rab7 recruits RILP on the late endosomal/lysosomal membrane and RILP then functions as a Rab7 effector controlling transport to degradative compartments. Indeed, RILP induces recruitment of dynein-dynactin motor complexes to Rab7-containing late endosomes and lysosomes. Recently, Rab7 and RILP have been found to be key proteins also for the biogenesis of phagolysosomes. Therefore, RILP represents probably an important factor for all endocytic routes to lysosomes. In this study, we show, using the yeast two-hybrid system, that RILP is able to interact with itself. The data obtained with the two-hybrid system were confirmed using co-immunoprecipitation in HeLa cells. The data together indicate that RILP, as already demonstrated for several other Rab effector proteins, is capable of self-association, thus probably forming a homo-dimer.
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http://dx.doi.org/10.1016/j.bbrc.2005.06.067DOI Listing
August 2005