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Sofia, Bulgaria

ProxAgen Ltd

Sofia, Bulgaria
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Amicosante M.,University of Rome Tor Vergata | Amicosante M.,ProxAgen Ltd. | Ciccozzi M.,Instituto Superiore Of Sanita
New Microbiologica | Year: 2010

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.

Minchev P.,Sofia University | Ciccozzi M.,Instituto Superiore Of Sanita | Amicosante M.,University of Rome Tor Vergata | Amicosante M.,ProxAgen Ltd.
New Microbiologica | Year: 2011

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.

Grifoni A.,University of Rome Tor Vergata | Montesano C.,University of Rome Tor Vergata | Salerno A.,University of Palermo | Colizzi V.,University of Rome Tor Vergata | And 2 more authors.
Molecular Immunology | Year: 2013

HLA class I molecules play a role in the regulation of innate immune response. Therefore, the interaction of HLA class I molecules with different activating and inhibitory receptors leads to balancing the immune response. Among the different family of receptors, NK receptors KIR3DL1/S1 and LIR1, play a major role. Aim of this study was to evaluate the role of amino acid polymorphic positions of HLA class I molecules interacting with NK receptors in HIV progression. In order to minimize the influence of viral variability, a cohort of children with a nosocomial monophyletic HIV-1 infection from the Benghazi Children Hospital has been evaluated. To assess the role of single amino acid positions, we translated all HLA alleles in the different amino acid position polymorphisms. Interestingly, the polymorphism Val 194 located in the α3-domain of HLA-B, resulted associated with LTNP (LTNP = 73.08%, FP = 34.78%; P<0.02). When Val is present at position 194, HLA-B is known to interact with the receptor LIR1 (ILT2/LILRB1/CD85j). Therefore, we analyzed the role of the polymorphism in position 194 in HLA-B/LIR1 interaction by homology molecular modeling. The change Val to Ile at position 194 alters significantly the network of interaction between the amino acid residues of HLA-B and LIR1. In conclusion, considering the limitation of the small population evaluated, polymorphisms outside the peptide binding region of the HLA class I molecule can play a key role in HIV progression through interaction with other immune-relevant receptors. © 2012 Elsevier Ltd.

Dimitrov V.,Regional Tuberculosis Dispensary | Saltini C.,University of Rome Tor Vergata | Amicosante M.,University of Rome Tor Vergata | Amicosante M.,ProxAgen Ltd.
Diagnostic Microbiology and Infectious Disease | Year: 2013

Current diagnostic standards for Mycobacterium tuberculosis (MTB) infection do not distinguish between active and latent tuberculosis (TB). To identify specific biomarkers characterizing the different forms of TB infection, we investigated in parallel with the QuantiFERON -TB Gold In-Tube (QFT-IT) the use of flow cytometry measuring CD4 and CD8 MTB-specific immune response in 17 active-TB patients, 21 health care workers (HCW), 14 recent contacts of TB patients (RC-TB), and 10 bacille Calmette Guerin (BCG)-vaccinated healthy controls (BCG-HC).A correlation (r = 0.4526, P = 0.0002) was found only between the amount of IFN-γ measured by QFT-IT and the frequency of CD4+/CD69+/IFN-γ+ T cells. The frequency of CD4+/CD69+/IFNγ+ responding T cells was higher in active-TB patients (0.254 ± 0.336%, P < 0.01) compared to the other groups. The response of QFT-IT antigen-specific CD8+/CD69+/IFNγ+ T cells was significantly higher in RC-TB (0.245 ± 0.305%, P < 0.05) compared to the other study groups. © 2013 Elsevier Inc.

Giovanetti M.,National Institute of Health | Grifoni A.,University of Rome Tor Vergata | Lo Presti A.,National Institute of Health | Cella E.,National Institute of Health | And 7 more authors.
Journal of Medical Virology | Year: 2015

Zaire Ebola virus (EBOV) is an enveloped non-segmented negative strand RNA virus of 19kb in length belonging to the family Filoviridae. The virus was isolated and identified in 1976 during the epidemic of hemorrhagic fever in Zaire. The most recent outbreak of EBOV among humans, was that occurred in the forested areas of south eastern Guinea, that began in February 2014 and is still ongoing. The recent Ebola outbreak, is affecting other countries in West Africa, in addiction to Guinea: Liberia, Nigeria, and Sierra Leone. In this article, a selective pressure analysis and homology modeling based on the G Glycoprotein (GP) sequences retrieved from public databases were used to investigate the genetic diversity and modification of antibody response in the recent outbreak of Ebola Virus. Structural and the evolutionary analysis underline the 2014 epidemic virus being under negative selective pressure does not change with respect to the old epidemic in terms of genome adaptation. © 2015 Wiley Periodicals, Inc.

PubMed | ProxAgen Ltd, National Institute of Health, University of Rome La Sapienza, University of Rome Tor Vergata and 2 more.
Type: Journal Article | Journal: Asian Pacific journal of tropical medicine | Year: 2016

To explore the genetic diversity and the modification of antibody response in the recent outbreak of Ebola Virus.Sequences retrieved from public databases, the selective pressure analysis and the homology modeling based on the all protein (nucleoprotein, VP35, VP40, soluble glycoprotein, small soluble glycoprotein, VP30, VP24 and polymerase) were used.Structural proteins VP24, VP30, VP35 and VP40 showed relative conserved sequences making them suitable target candidates for antiviral treatment. On the contrary, nucleoprotein, polymerase and soluble glycoprotein have high mutation frequency.Data from this study point out important aspects of Ebola virus sequence variability that for epitope and vaccine design should be considered for appropriate targeting of conserved protein regions.

Grifoni A.,University of Rome Tor Vergata | Montesano C.,University of Rome Tor Vergata | Palma P.,Bambino Gesu Childrens Hospital | Giovannetti M.,Instituto Superiore Of Sanita | And 9 more authors.
Immunology | Year: 2014

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.

Crescenzi A.,Biomedical University of Rome | Annibali O.,Biomedical University of Rome | Bianchi A.,Biomedical University of Rome | Pagano A.,Biomedical University of Rome | And 3 more authors.
Leukemia Research | Year: 2016

Multiple myeloma patients may develop extraosseous involvement in the course of the disease making prognosis very poor and new drugs clearly needed. The PD-1/PD-L1 axis has emerged as a master immune checkpoint in antitumor responses and recent studies investigated the role of PD-L1 in multiple myeloma cells; no data however are still available about PD-L1 expression in extramedullary localizations. We demonstrate PD-L1 expression in 4/12 cases of extraosseous myeloma suggesting that these lesions represent a specialized microenvironment. We found presence of PD-1+ infiltrating lymphocytes in all observed cases supporting the relevance of PD-1/PD-L1 checkpoint in extramedullary myeloma. We also investigated the correlation in PD1/PD-L1 staining between marrow staining and EMP lesions. © 2016

Piubelli L.,University of Insubria | Piubelli L.,Polytechnic of Milan | Campa M.,University of Insubria | Campa M.,Foundation Edmund Machinery | And 10 more authors.
Microbial Cell Factories | Year: 2013

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.

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 | Year: 2016

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.

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