Francesca Bugli

Human Monoclonal Antibody-Based Therapy in the Treatment of Invasive Candidiasis

Invasive candidiasis (IC) represents the leading fungal infection of humans causing life-threatening disease in immunosuppressed and neutropenic individuals including also the intensive care unit patients. Despite progress in recent years in drugs development for the treatment of IC, morbidity and mortality rates still remain very high. Historically, cell-mediated immunity and innate immunity are considered to be the most important lines of defense against candidiasis. Nevertheless recent evidence demonstrates that antibodies with defined specificities could act with different degrees showing protection against systemic and mucosal candidiasis. Mycograb is a human recombinant monoclonal antibody against heat shock protein 90 (Hsp90) that was revealed to have synergy when combined with fluconazole, caspofungin, and amphotericin B against a broad spectrum of Candida species. Furthermore, recent studies have established an important role for Hsp90 in mediating Candida resistance to echinocandins, giving to this antibody molecule even more attractive biological properties. In response to the failure of marketing authorization by the CHMP (Committee for Medicinal Products for Human Use) a new formulation of Mycograb, named Mycograb C28Y variant, with an amino acid substitution was developed in recent years. First data on Mycograb C28Y variant indicate that this monoclonal antibody lacked efficacy in a murine candidiasis model.

Mapping B-cell epitopes of hepatitis C virus E2 glycoprotein using human monoclonal antibodies from phage display libraries.

ABSTRACT Clinical and experimental evidence indicates that the hepatitis C virus (HCV) E2 glycoprotein (HCV/E2) is the most promising candidate for the development of an effective anti-HCV vaccine. Identification of the human epitopes that are conserved among isolates and are able to elicit protective antibodies would constitute a significant step forward. This work describes the mapping of the B-cell epitopes present on the surface of HCV/E2, as recognized by the immune system during infection, by the analysis of the reciprocal interactions of a panel of human recombinant Fabs derived from an HCV-infected patient. Three unrelated epitopes recognized by antibodies with no neutralization-of-binding (NOB) activity were identified; a fourth, major epitope was defined as a clustering of minor epitopes recognized by Fabs endowed with strong NOB activity.

Biomimetic antimicrobial cloak by graphene-oxide agar hydrogel

Antibacterial surfaces have an enormous economic and social impact on the worldwide technological fight against diseases. However, bacteria develop resistance and coatings are often not uniform and not stable in time. The challenge is finding an antibacterial coating that is biocompatible, cost-effective, not toxic, and spreadable over large and irregular surfaces. Here we demonstrate an antibacterial cloak by laser printing of graphene oxide hydrogels mimicking the Cancer Pagurus carapace. We observe up to 90% reduction of bacteria cells. This cloak exploits natural surface patterns evolved to resist to microorganisms infection, and the antimicrobial efficacy of graphene oxide. Cell integrity analysis by scanning electron microscopy and nucleic acids release show bacteriostatic and bactericidal effect. Nucleic acids release demonstrates microorganism cutting, and microscopy reveals cells wrapped by the laser treated gel. A theoretical active matter model confirms our findings. The employment of biomimetic graphene oxide gels opens unique possibilities to decrease infections in biomedical applications and chirurgical equipment; our antibiotic-free approach, based on the geometric reduction of microbial adhesion and the mechanical action of Graphene Oxide sheets, is potentially not affected by bacterial resistance.

A vector for the expression of recombinant monoclonal Fab fragments in bacteria

The availability of genes coding for monoclonal Fab fragments of a desired specificity permits their expression in bacteria and provides a simple method for the generation of good quality reagents. In this paper we describe a new phagemid vector for the production of recombinant Fabs from genes obtained from phage display combinatorial libraries. The phagemid features an antibiotic resistance cassette which, once inserted between the heavy chain fragment and the light chain genes, avoids unwanted recombination and preserves useful restriction sites not affecting the Fab production rate.

In Vitro Interaction between Alginate Lyase and Amphotericin B against Aspergillus fumigatus Biofilm Determined by Different Methods

ABSTRACT Aspergillus fumigatus biofilms represent a problematic clinical entity, especially because of their recalcitrance to antifungal drugs, which poses a number of therapeutic implications for invasive aspergillosis, the most difficult-to-treat Aspergillus-related disease. While the antibiofilm activities of amphotericin B (AMB) deoxycholate and its lipid formulations (e.g., liposomal AMB [LAMB]) are well documented, the effectiveness of these drugs in combination with nonantifungal agents is poorly understood. In the present study, in vitro interactions between polyene antifungals (AMB and LAMB) and alginate lyase (AlgL), an enzyme degrading the polysaccharides produced as extracellular polymeric substances (EPSs) within the biofilm matrix, against A. fumigatus biofilms were evaluated by using the checkerboard microdilution and the time-kill assays. Furthermore, atomic force microscopy (AFM) was used to image and quantify the effects of AlgL-antifungal combinations on biofilm-growing hyphal cells. On the basis of fractional inhibitory concentration index values, synergy was found between both AMB formulations and AlgL, and this finding was also confirmed by the time-kill test. Finally, AFM analysis showed that when A. fumigatus biofilms were treated with AlgL or polyene alone, as well as with their combination, both a reduction of hyphal thicknesses and an increase of adhesive forces were observed compared to the findings for untreated controls, probably owing to the different action by the enzyme or the antifungal compounds. Interestingly, marked physical changes were noticed in A. fumigatus biofilms exposed to the AlgL-antifungal combinations compared with the physical characteristics detected after exposure to the antifungals alone, indicating that AlgL may enhance the antibiofilm activity of both AMB and LAMB, perhaps by disrupting the hypha-embedding EPSs and thus facilitating the drugs to reach biofilm cells. Taken together, our results suggest that a combination of AlgL and a polyene antifungal may prove to be a new therapeutic strategy for invasive aspergillosis, while reinforcing the EPS as a valuable antibiofilm drug target.

Human DDX3 protein is a valuable target to develop broad spectrum antiviral agents.

Significance Human DEAD-box polypeptide 3 (DDX3) is an ATPase/RNA helicase involved in the replication of many viral pathogens. We reported herein the first inhibitor, to our knowledge, of the helicase binding site of DDX3 endowed with a broad spectrum antiviral activity [HIV-1 WT, HIV drug-resistant strains, Hepatitis C virus (HCV), Dengue virus (DENV), and West Nile virus (WNV)]. The good toxicity profile suggests that the DDX3 activity, although essential for viruses, could be dispensable to the cells, validating DDX3 as a pharmaceutical target. Our results clearly showed that DDX3 inhibitors could be exploited to treat HIV/HCV coinfections, emerging infectious diseases (such as DENV and WNV), and HIV-1 patients carrying drug-resistant strains. Each of these three medical conditions currently represents a major challenge for clinical treatment. Targeting a host factor essential for the replication of different viruses but not for the cells offers a higher genetic barrier to the development of resistance, may simplify therapy regimens for coinfections, and facilitates management of emerging viral diseases. DEAD-box polypeptide 3 (DDX3) is a human host factor required for the replication of several DNA and RNA viruses, including some of the most challenging human pathogens currently circulating, such as HIV-1, Hepatitis C virus, Dengue virus, and West Nile virus. Herein, we showed for the first time, to our knowledge, that the inhibition of DDX3 by a small molecule could be successfully exploited for the development of a broad spectrum antiviral agent. In addition to the multiple antiviral activities, hit compound 16d retained full activity against drug-resistant HIV-1 strains in the absence of cellular toxicity. Pharmacokinetics and toxicity studies in rats confirmed a good safety profile and bioavailability of 16d. Thus, DDX3 is here validated as a valuable therapeutic target.

Helicobacter pylori infection contributes to placental impairment in preeclampsia: basic and clinical evidences.

Preeclampsia (PE) is a major cause of maternal and neonatal morbidity and mortality. Epidemiological association between Helicobacter pylori (Hp) infection and PE onset has been widely shown. The aim of this study was to analyze a possible correlation between Hp infection and the severity of clinical presentation of PE and to identify an immunologic mechanism triggered by Hp infection potentially contributing to the pathogenesis of PE.

The PavA-like Fibronectin-Binding Protein of Enterococcus faecalis, EfbA, Is Important for Virulence in a Mouse Model of Ascending Urinary Tract Infection

Enterococcus faecalis is an established nosocomial pathogen, yet the pathogenesis of enterococcal infections, particularly of urinary tract infections (UTIs), remains to be fully elucidated. Fibronectin-binding proteins have been identified as potent adhesins in pathogenic Gram-positive cocci. Here, we characterized EfbA, which is encoded by the enterococcal orthologue of Streptococcus pneumoniae pavA. Similar to PavA, the anchorless EfbA protein was localized to the enterococcal cell outer surface and bound to immobilized human fibronectin. In addition to abrogated EfbA expression, deletion of the efbA gene eliminated EfbA from the cell surface and drastically reduced the enterococcal cell binding to immobilized fibronectin. The ΔefbA deletion mutant was highly attenuated vs wild-type in a murine ascending UTI model, consistent with an increased tropism for the kidney relative to the bladder. These results provide the first evidence that EfbA of E. faecalis plays a role in UTIs, probably contributing to the pathogenesis in this site.

Production and Characterization of a Human Recombinant Monoclonal Fab Fragment Specific for Influenza A Viruses

ABSTRACT A human recombinant monoclonal Fab fragment that specifically recognizes all the influenza A virus strains tested was produced in transformed Escherichia coli using the phage display technique. No strain of influenza B virus reacted with it. It was purified after four cycles of panning and by a single passage through an immunoaffinity column. About 1 mg of pure monoclonal antibody was obtained from 1 liter of culture medium in 3 working days. The Fab fragment reacted with a viral 27-kDa protein, which could reasonably be a matrix protein. Indirect immunofluorescence tests performed on virus-infected MDCK cells showed that this Fab fragment was at least equally efficient as other commercial monoclonal antibody-based systems in detecting influenza A viral infections. The potential advantages of human recombinant Fabs on murine monoclonal antibodies are discussed.

Analysis of heat-induced changes in protein expression of Stenotrophomonas maltophilia K279a reveals a role for GroEL in the host-temperature adaptation

Stenotrophomonas maltophilia is a microorganism of environmental and clinical importance as well as a frequent airway colonizer of cystic fibrosis (CF) individuals. We combined 2-DE and MALDI-TOF MS to profile the protein expression in S. maltophilia K279a, a completely sequenced clinical isolate, grown at 37 °C with respect to the strain grown at 26 °C. Among the proteins up-regulated at 37 °C, we identified GroEL, a molecular chaperone that mainly assist the folding and unfolding of proteins under both normal and stress conditions. A 2.4-kb groESL mRNA was detected independently by Northern blot analyses with a groES- and a groEL-specific probe, indicating that S. maltophilia groES and groEL form an operon. Primer extension analysis of S. maltophilia groESL done in Escherichia coli showed that 2 promoters, Pσ(32) and Pσ(70), were utilized under the heat-shock and normal condition, respectively, whereas S. maltophilia groEL was shown to act as a heat-shock gene at 37 °C, 42 °C, and, to a lesser extent, at 50 °C by real-time RT-PCR analyses. Finally, immunoblot analyses revealed that S. maltophilia GroEL strongly reacted with sera from CF patients chronically infected by the microorganism, but did not with sera from CF patients with sporadic infection or uninfected.