Lactoferrin, a member of the transferrin family of approximately 80 kDa, consists of a single polypeptide chain folded in two symmetric, globular lobes (N- and C-lobes), each able to bind one ferric ion. This glycoprotein, found in physiological fluids of mammals, plays an important role in immune regulation and in defense mechanisms against bacteria, fungi, parasites, and viruses. Although the antiviral activity of lactoferrin is one of the major biological functions of such protein, the mechanism of action is still under debate. We have investigated both the role of tryptic fragments of bovine lactoferrin and the mechanism of lactoferrin antiviral effect toward adenovirus infection in HEp-2 cells. The results obtained demonstrated that the anti-adenovirus activity of lactoferrin is mediated by the N-terminal half of the protein as the N-lobe was able to inhibit adenovirus infection, even if at lower extent than undigested lactoferrin, whereas C-lobe was ineffective. The results also showed that the anti-adenovirus action of lactoferrin and of its N-terminal peptide lactoferricin took place on virus attachment to cell membrane, mainly through competition for common glycosaminoglycan receptors. The data provide evidence that the anti-adenovirus activity of lactoferrin is mediated mainly by the cluster of positive charges at the N-terminus of whole molecule and that the N-terminal peptide lactoferricin alone is sufficient to prevent infection.

Bovine lactoferrin (bLf) is a multifunctional glycoprotein that plays an important role in innate immunity against infections, including influenza. Here we have dissected bLf into its C-and N-lobes and show that inhibition of influenza virus hemagglutination and cell infection is entirely attributable to the C-lobe and that all major virus subtypes, including H1N1 and H3N2, are inhibited. By far-western blotting and sequencing studies, we demonstrate that bLf C-lobe strongly binds to the HA 2 region of viral hemagglutinin, precisely the highly conserved region containing the fusion peptide. By molecular docking studies, three C-lobe fragments were identified which inhibited virus hemagglutination and infection at fentomolar concentration range. Besides contributing to explain the broad anti-influenza activity of bLf, our findings lay the foundations for exploiting bLf fragments as source of potential anti-influenza therapeutics.

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We recently demonstrated that lactoferrin, an antimicrobial glycoprotein, can inhibit adenovirus infection by competing for common glycosaminoglycan receptors. This study further characterizes the antiadenovirus activity of the protein, thus demonstrating that lactoferrin neutralizes infection by binding to adenovirus particles and that its targets are viral III and IIIa structural polypeptides.Lactoferrin is a multifunctional glycoprotein that possesses a variety of physiological roles, such as promotion of iron absorption (26), immunomodulation (16), and inhibiting activity toward different pathogens (14,16,24,26,(32)(33)(34), including viruses (1, 2, 7, 9-13, 17-19, 21, 25, 28-30, 38). For all viruses investigated to date, lactoferrin exerts its antiviral activity during the early phases of infection. Recently, we demonstrated a competition between bovine lactoferrin (bLf) and adenovirus for the attachment to cell membrane glycosaminoglycans, even though a binding between lactoferrin and viral particles could not be ruled out (6). The present study provides deeper insight into the mechanism of the antiadenoviral action of bLf, demonstrating that this glycoprotein also exerts its antiadenoviral activity by a direct interaction with adenovirus particles.Human epidermoid carcinoma larynx (HEp-2) cells, obtained from the American Type Culture Collection (ATCC), and human adenovirus type 2 were grown as previously described (2). Viral particles were purified according to the methods of Wadell and coworkers (35).bLf (Morinaga Milk Industry) and heparin (Sigma, 170 USP U/mg) were dissolved in phosphate-buffered saline (PBS; pH 7.2). bLf was biotinylated according to the manufacturer's instructions (Amersham). Dot blot assays were carried out by applying 50 l of purified adenovirus, heparin, or PBS to nitrocellulose paper and using a Bio-Dot apparatus according to the manufacturer's instructions (Bio-Rad). Air-dried membranes were blocked in block solution, washed with PBS containing 0.1% Tween 20, and then incubated with biotinylated bLf. After washings in PBS containing 0.1% Tween 20, streptavidin-horseradish peroxidase (streptavidin-HRP) conjugate was added to the blot. Blot was revealed by an enhanced chemiluminescence kit (Amersham Biosciences) and the 3,3Ј,5,5Ј-tetramethylbenzidine (TMB) substrate kit for peroxidase (Vector Laboratories) according to the manufacturer's instructions.Neutralization of adenovirus binding to cells was carried out by incubating lactoferrin with adenovirus for 1 h at 37 or 4°C.The suspensions were then added to cells for 1 h at 4°C. Viral antigen synthesis was monitored 24 h after infection (37°C) by immunofluorescence as previously described (2). Statistical analysis was performed using the Student's t test for unpaired data. P values of Ͻ0.05 were considered significant. Adenovirus proteins were resolved by sodium dodecyl sulfate-10% polyacrylamide gel electrophoresis (SDS-PAGE) (15) under denaturing conditions as described by Wadell and coworkers (36). Coxsackievirus proteins were...

It is well known that lactoferrin (Lf) is a potent inhibitor towards several enveloped and naked viruses, such as rotavirus, enterovirus and adenovirus. Lf is resistant to tryptic digestion and breast-fed infants excrete high levels of faecal Lf, so that its effect on viruses replicating in the gastrointestinal tract is of great interest. In this report, we analysed the mechanism of the antiviral action of this protein in three viral models which, despite representing different genoma and replication strategies, share the ability to infect the gut. Concerning the mechanism of action against rotavirus, Lf from bovine milk (BLf) possesses a dual role, preventing virus attachment to intestinal cells by binding to viral particles, and inhibiting a post adsorption step. The BLf effect towards poliovirus is due to the interference with an early infection step but, when the BLf molecule is saturated with Zn+2 ions, it is also capable of inhibiting viral replication after the viral adsorption phase. The anti-adenovirus action of BLf takes place on virus attachment to cell membranes through competition for common glycosaminoglycan receptors and a specific interaction with viral structural polypeptides. Taken together, these findings provide further evidence that Lf is an excellent candidate in the search of natural agents against viral enteric diseases, as it mainly acts by hindering adsorption and internalisation into cells through specific binding to cell receptors and/or viral particles.

Influenza is one of the main plagues worldwide. The statistical likelihood of a new pandemic outbreak, together with the alarming emergence of influenza virus strains that are resistant to available antiviral medications, highlights the need for new antiviral drugs. Lactoferrin, a 80 kDa bi-globular iron-binding glycoprotein, is a pleiotropic factor with potent antimicrobial and immunomodulatory activities. Although the antiviral effect of lactoferrin is one of its major biological functions, the mechanism of action is still under debate. In this research, we have analyzed the effect of bovine lactoferrin (bLf) on Influenza A virus infection in vitro. Our results showed that (i) Influenza virus infected cells died as a result of apoptosis, (ii) bLf treatment inhibited programmed cell death by interfering with function of caspase 3, a major virus-induced apoptosis effector, and (iii) bLf efficiently blocked nuclear export of viral ribonucleoproteins so preventing viral assembly. These results provide further insights on the antiviral activity of bLf and suggest novel strategies for treatment of Influenza virus infection.

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Schistosomiasis is the most significant neglected tropical parasitic disease caused by helminths in terms of morbidity and mortality caused by helminths. In this work, we present the antischistosomal activity against Schistosoma mansoni of a rationally selected small set of thiazinoquinone derivatives, some of which were previously found to be active against Plasmodium falciparum and others synthesized ad hoc. The effects on larvae, juvenile, and adult parasite viability as well as on egg production and development were investigated, resulting in the identification of new multistage antischistosomal hit compounds. The most promising compounds 6, 8, 13, and 14 with a LC 50 value on schistosomula from ∼5 to ∼15 μM also induced complete death of juvenile (28 days old) and adult worm pairs (7 weeks old) and a detrimental effect on egg production and development in vitro. Structure−activity relationships (SARs) were analyzed by means of computational studies leading to the hypothesis of a redox-based mechanism of action with a one-electron reduction bioactivation step and the subsequent formation of a toxic semiquinone species, similarly to what was previously observed for the antiplasmodial activity. Our results also evidenced that the selective toxicity against mammalian cells or parasites as well as specific developmental stages of a parasite can be addressed by varying the nature of the introduced substituents.