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Sofia, Bulgaria

Minchev P.,Sofia University | Ciccozzi M.,Parasitic and Immunomediated Disease | Amicosante M.,University of Rome Tor Vergata | Amicosante M.,ProxAgen Ltd.
New Microbiologica

QuantiFERON-TB data from 50 children with tuberculosis were analysed to evaluate age related effects. Significantly higher IFN-y responses to TB-specific antigens were associated with younger age, but no difference was found with Mitogen responses. Extrapolating IGRA responses to a Mitogen does not reflect those induced by an antigen-specific stimulus. QFT-IT responses to TB-specific antigens are not compromised with young age. Source

Amicosante M.,University of Rome Tor Vergata | Amicosante M.,ProxAgen Ltd. | Ciccozzi M.,Parasitic and Immunomediated Disease
New Microbiologica

Tuberculosis (TB) remains a public health challenge and its control requires the use efficient diagnostic tools. Mycobacterium tubercubsis (MTB) elicits a strong immune response upon infection, a phenomenon measured by the old tuberculin skin test (TST). However, this test has many limitations and a high rate of positivity in BCG-vaccinated subjects. Recent studies have identified several MTB-antigens for diagnostic use, including the ESAT-6 and CFP-10 proteins. Based on these antigens, one of the most significant developments in the diagnostic armamentarium for TB has been the assays based on IFN- determination (IGRAs). The assays stem from the principle that T-cells of infected individuals produce IFN-gamma when they re-encounter the MTB antigens in vitro and this can be measured by a conventional ELISA test. The evaluation of IGRAs in different clinical settings showed many advantages over TST. The worldwide diffusion of IGRAs has increased the knowledge on their clinical use and a number of guidelines have been devised for their application. The two-step approach (first using TST followed by IGRA for confirmation) is the most favored strategy for IGRA-use in the general population, while the use of IGRAs alone is suggested in particular clinical settings and/or patient groups. Even if these tests are still costly there are a number of cost effective advantages in the "targeted" use of IGRAs over the TST. The work we present summarises all these aspects. Source

Piubelli L.,University of Insubria | Piubelli L.,Polytechnic of Milan | Campa M.,University of Insubria | Temporini C.,University of Pavia | And 9 more authors.
Microbial Cell Factories

Background: A number of valuable candidates as tuberculosis vaccine have been reported, some of which have already entered clinical trials. The new vaccines, especially subunit vaccines, need multiple administrations in order to maintain adequate life-long immune memory: this demands for high production levels and degree of purity.Results: In this study, TB10.4, Ag85B and a TB10.4-Ag85B chimeric protein (here-after referred as full) - immunodominant antigens of Mycobacterium tuberculosis - were expressed in Escherichia coli and purified to homogeneity. The rational design of expression constructs and optimization of fermentation and purification conditions allowed a marked increase in solubility and yield of the recombinant antigens. Indeed, scaling up of the process guaranteed mass production of all these three antigens (2.5-25 mg of pure protein/L cultivation broth). Quality of produced soluble proteins was evaluated both by mass spectrometry to assess the purity of final preparations, and by circular dichroism spectroscopy to ascertain the protein conformation. Immunological tests of the different protein products demonstrated that when TB10.4 was fused to Ag85B, the chimeric protein was more immunoreactive than either of the immunogenic protein alone.Conclusions: We reached the goal of purifying large quantities of soluble antigens effective in generating immunological response against M. tuberculosis by a robust, controlled, scalable and economically feasible production process. © 2013 Piubelli et al.; licensee BioMed Central Ltd. Source

Bavaro T.,University of Pavia | Piubelli L.,University of Insubria | Piubelli L.,Polytechnic of Milan | Amicosante M.,University of Rome Tor Vergata | And 2 more authors.
Current Organic Chemistry

The most effective approach in controlling infectious diseases and other immune related disorders has been the development and the extensive use of preventive vaccines and vaccine therapies. However, we still need effective vaccines against some of the most threatening infectious agents. The standard costs and approaches in developing and producing a vaccine are dramatically high. In the recent past, the common effort of scientists resulted in novel approaches to vaccine target identification largely based on bioinformatics, immunoinformatics and structural biology, reducing time to identification of relevant antigens. These strategies, together with an increased knowledge of host-pathogen interactions and protection mechanisms, is enhancing the rapid development of novel vaccines. The reverse vaccinology approach also allowed the development of a large number of new recombinant protein-based vaccines. However, this approach results poorly efficient against genetically complex diseases, such as malaria, tuberculosis, HIV, and towards cancer. In the latter cases, the modern approach for designing efficient vaccines is moving to structure vaccinology. The deep characterization of different epitopes permits the rational design of new immunogenic products. For example, new products obtained by combination of differently defined antigens, such as chimeric proteins, were proposed as improved vaccines against tuberculosis. Similarly, the structural characterization of antigenic oligosaccharides allowed the development of a commercial vaccine for the prevention of Haemophilus influenzae type B. This approach has also been proposed for the treatment of other infectious diseases, such as meningococcal infections, pneumococcal infections and tuberculosis as well as for the treatment of certain types of cancer. © 2016 Bentham Science Publishers. Source

Grifoni A.,University of Rome Tor Vergata | Montesano C.,University of Rome Tor Vergata | Palma P.,Bambino Gesu Childrens Hospital | Giovannetti M.,Istituto Superiore di Sanita | And 9 more authors.

Summary: Viral and host factors can influence HIV-1 progression, among them human leucocyte antigen (HLA) has shown the strongest effect. However, studies on the functional contribution of HLA in controlling HIV progression toward AIDS are limited by multiple issues, including the viral strain variability within the study subjects. In this study, in a cohort of children infected with a monophyletic strain (CRF02_AG) during an outbreak, we evaluated the HIV-1 Gag, Vif, Vpr, Tat and hepatitis C virus E1/E2 (as control) proteins circulating in a cohort for the capability to be presented by the HLA molecules in the same population. A total of 70 Non-progressors and 37 Progressors to AIDS were evaluated. In the presence of a constant capability of HIV-1 to mutate in the region containing epitopes of Gag protein, the number of epitopes recognized in silico by the combination of the HLA alleles along the Gag consensus sequence is significantly higher in the Non-progressors compared with Progressors (HLA-A: Non-progressors = 1·532 ± 1·211, Progressors = 0·7714± 1·031, P = 0·0016; HLA-B: Non-progressors = 1·556 ± 1·298, Progressors = 1·000 ± 0·817, P = 0·0319; HLA-DR: Non-progressors = 13·30± 9·488, Progressors = 7·294 ± 6·952, P = 0·0006). Similar results were obtained for the other HIV-1 proteins Vif and Vpr, whereas no differences were obtained in the number of epitopes for the hepatitis C virus E1/E2 protein sequence or for the scrambled HIV-1 sequence. Finally, the results were confirmed also in a subgroup of subjects where both HLA typing and Gag sequence were available. In conclusion, in the absence of bias due to viral strain diversity, it is the overall fitting of the HLA molecules that are capable of better binding HIV-1 proteins in determining the major role in the control of HIV-1 replication and progression to AIDS. © 2014 John Wiley & Sons Ltd. Source

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