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David Lembo

Università degli Studi di Torino, Deparment of Clinical and Biological Sciences, Faculty Member

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More than 25 years experience in Virology with focus on virus-cell interactions and research and development of antivirals.

I am currently heading the Laboratory of Molecular Virology an Antiviral Research at the University of Turin, Italy

My biographical details have been included in the 2011 and 2012 Edition of "Marquis Who's Who in the World".

I am the author or co-author of more than 70 publications in peer-reviewed international journals.

I am inventor of several patents in the field of antivirals and nanomedicine.

In 2005 I was awarded the “Technology Scouting in Academia” prize.

I am a scientific consultant of several biotech companies.

I am a co-founder of the biotech company RotaLactis.

I am a member of the Editorial Board of Scientific Reports (Nature publishing Group)

Research Interests
• Studies on virus-cell interactions.
• Research and development of antiviral molecules with a focus on herpesviruses,
papillomaviruses, rotaviruses rhinoviruses and respiratory syncytial virus.
• Research and development of innovative formulations based on nanocarriers for the delivery of
antiviral and antibacterial drugs, peptides, enzymes and nucleic acids.
• Ethnomedicine

Specialties: Viruses routinely screened for antiviral drug discovery in my lab:
- Herpes simplex virus
- Cytomegalovirus
- Human papillomaviruses
- Rotaviruses
- Respiratory syncytial virus
- Rhinoviruses

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Papers by David Lembo

Identification of Equine Lactadherin-Derived Peptides that Inhibit Rotavirus Infection via Integrin Receptor Competition

by David Lembo, Barbara Coulson, and Amedeo Conti

The Journal of biological chemistry, Jan 26, 2015

Human rotavirus is the leading cause of severe gastroenteritis in infants and children under the ... more Human rotavirus is the leading cause of severe gastroenteritis in infants and children under the age of five years in both developed and developing countries. Human lactadherin, a milk-fat globule membrane (MFGM) glycoprotein, inhibits human rotavirus infection in vitro, whereas bovine lactadherin is not active. Moreover, it protects breastfed infants against symptomatic rotavirus infections. To explore the potential antiviral activity of lactadherin sourced by equines we undertook a proteomic analysis of MFGM proteins from donkey milk and elucidated its amino acid sequence. Alignment of the human, bovine and donkey lactadherin sequences revealed the presence of an Asp-Gly-Glu (DGE) α2β1 integrin-binding motif in the N-terminal domain of donkey sequence only. Since integrin α2β1 plays a critical role during early steps of rotavirus host cell adhesion, we tested a mini-library of donkey lactadherin-derived peptides containing DGE sequence for anti-rotavirus activity. A 20 amino acid ...

Early inhibitors of human cytomegalovirus: State-of-art and therapeutic perspectives

by Beatrice Mercorelli and David Lembo

Pharmacology & Therapeutics, 2011

Human cytomegalovirus (HCMV) infection is associated with severe morbidity and mortality in immun... more Human cytomegalovirus (HCMV) infection is associated with severe morbidity and mortality in immunocompromised individuals, mainly transplant recipients and AIDS patients, and is the most frequent cause of congenital malformations in newborn children. To date, few drugs are licensed for the treatment of HCMV infections, most of which target the viral DNA polymerase and suffer from many drawbacks, including long-term toxicity, low potency, and poor bioavailability. In addition, the emergence of drug-resistant viral strains is becoming an increasing problem for disease management. Finally, none of the current anti-HCMV drugs have been approved for the treatment of congenital infections. For all these reasons, there is still a strong need for new anti-HCMV drugs with novel mechanisms of action. The first events of the virus replication cycle, including attachment, entry, immediate-early gene expression, and immediate-early functions-in particular that of Immediate-Early 2 protein-represent attractive targets for the development of novel antiviral compounds. Such inhibitors would block not only the expression of viral immediate-early proteins, which play a key role in the pathogenesis of HCMV infection, but also the host immunomodulation and the changes to cell physiology induced by the first events of virus infection. This review describes the current knowledge on the initial phases of HCMV replication, their validation as potential novel antiviral targets, and the development of compounds that block such processes.

Hexagonal-shaped chondroitin sulfate self-assemblies have exalted anti-HSV-2 activity

Carbohydrate Polymers, 2015

The initial step in mucosal infection by the herpes simplex virus type 2 (HSV-2) requires its bin... more The initial step in mucosal infection by the herpes simplex virus type 2 (HSV-2) requires its binding to certain glycosaminoglycans naturally present on host cell membranes. We took advantage of this interaction to design biomimetic supramolecular hexagonal-shaped nanoassemblies composed of chondroitin sulfate having exalted anti-HSV-2 activity in comparison with native chondroitin sulfate. Nanoassemblies were formed by mixing hydrophobically-modified chondroitin sulfate with α-cyclodextrin in water. Optimization of alkyl chain length grafted on chondroitin sulfate and the ratio between hydrophobically-modified chondroitin sulfate and α-cyclodextrin showed that more cohesive and well-structured nanoassemblies were obtained using higher α-cyclodextrin concentration and longer alkyl chain lengths. A structure-activity relationship was found between anti-HSV-2 activity and the amphiphilic nature of hydrophobically-modified chondroitin sulfate. Also, antiviral activity of hexagonal nanoassemblies against HSV-2 was further improved in comparison with hydrophobically-modified chondroitin sulfate. This work suggests a new biomimetic formulation approach that can be extended to other heparan-sulfate-dependent viruses.

Cytomegalovirus stimulates the expression of cellular enzymes involved in nucleotides biosynthesis

Journal of Clinical Virology, 1999

The AGMA1 poly(amidoamine) inhibits the infectivity of herpes simplex virus in cell lines, in human cervicovaginal histocultures, and in vaginally infected mice

Biomaterials, 2016

The development of topical microbicides is a valid approach to protect the genital mucosa from se... more The development of topical microbicides is a valid approach to protect the genital mucosa from sexually transmitted infections that cannot be contained with effective vaccination, like HSV and HIV infections. A suitable target of microbicides is the interaction between viral proteins and cell surface heparan sulfate proteoglycans (HSPGs). AGMA1 is a prevailingly cationic agmatine-containing polyamidoamine polymer previously shown to inhibit HSPGs dependent viruses, including HSV-1, HSV-2, and HPV-16. The aim of this study was to elucidate the mechanism of action of AGMA1 against HSV infection and assess its antiviral efficacy and biocompatibility in preclinical models. The results show AGMA1 to be a non-toxic inhibitor of HSV infectivity in cell cultures and human cervicovaginal histocultures. Moreover, it significantly reduced the burden of infection of HSV-2 genital infection in mice. The investigation of the mechanism of action revealed that AGMA1 reduces cells susceptibility to virus infection by binding to cell surface HSPGs thereby preventing HSV attachment. This study indicates that AGMA1 is a promising candidate for the development of a topical microbicide to prevent sexually transmitted HSV infections.

Novel Antipathogenic Peptides

The human cytomegalovirus

Pharmacology Therapeutics, Jun 1, 2003

Human cytomegalovirus (HCMV), a betaherpesvirus, represents the major infectious cause of birth d... more Human cytomegalovirus (HCMV), a betaherpesvirus, represents the major infectious cause of birth defects, as well as an important pathogen for immunocompromised individuals. The viral nucleocapsid containing a linear double-stranded DNA of 230 kb is surrounded by a proteinaceous tegument, which is itself enclosed by a loosely applied lipid bilayer. Expression of the HCMV genome is controlled by a cascade of transcriptional events that leads to the synthesis of three categories of viral proteins designated as immediate-early, early, and late. Clinical manifestations can be seen following primary infection, reinfection, or reactivation. About 10% of infants are infected by the age of 6 months following transmission from their mothers via the placenta, during delivery, or by breastfeeding. HCMV is a significant post-allograft pathogen and contributes to graft loss independently from graft rejection. Histopathologic examination of necropsy tissues demonstrates that the virus enters via the epithelium of the upper alimentary, respiratory, or genitourinary tracts. Hematogenous spreading is typically followed by infection of ductal epithelial cells. Infections are kept under control by the immune system. However, total HCMV clearance is rarely achieved, and the viral genome remains at selected sites in a latent state. Virological and molecular detection of HCMV, as well as serological demonstration of a specific immune response, are used for diagnosis. Treatment of HCMV infections is difficult because there are few options. The presently available drugs produced a significant clinical improvement, but suffer from poor oral bioavailability, low potency, development of resistance in clinical practice, and dose-limiting toxicities. D

The L1 protein of human papilloma virus 16 expressed by a fowlpox virus recombinant can assemble into virus-like particles in mammalian cell lines but elicits a non-neutralising humoral response

Antiviral Research, 2015

Human papilloma virus (HPV)-16 is the prevalent genotype associated with cervical tumours. Virus-... more Human papilloma virus (HPV)-16 is the prevalent genotype associated with cervical tumours. Virus-likeparticle (VLP)-based vaccines have proven to be effective in limiting new infections of high-risk HPVs, but their high cost has hampered their use, especially in the poor developing countries. Avipox-based recombinants are replication-restricted to avian species and represent efficient and safe vectors also for immunocompromised hosts, as they can elicit a complete immune response. A new fowlpox virus recombinant encoding HPV-L1 (FP L1 ) was engineered and evaluated side-by-side with a FP recombinant co-expressing L1 and green fluorescent protein (FP L1GFP ) for correct expression of L1 in vitro in different cell lines, as confirmed by Western blotting, immunofluorescence, real-time PCR, and electron microscopy. Mice were also immunised to determine its immunogenicity. Here, we demonstrate that the FP L1 recombinant better expresses L1 in the absence of GFP, correctly assembles structured capsomers into VLPs, and elicits an immune response in a preclinical animal model. To our knowledge, this is the first report of HPV VLPs assembled in eukaryotic cells using an avipox recombinant.

Cyclodextrin based nanosponges encapsulating camptothecin

Camptothecin (CAM), a plant alkaloid and a potent antitumor agent, has a limited therapeutic util... more Camptothecin (CAM), a plant alkaloid and a potent antitumor agent, has a limited therapeutic utility because of its poor aqueous solubility, lactone ring instability and serious side effects. Cyclodextrin-based nanosponges (NS) are a novel class of cross-linked derivatives of cyclodextrins. They have been used to increase the solubility of poorly soluble actives, to protect the labile groups and control the release. This study aimed at formulating complexes of CAM with three types of b-cyclodextrin NS obtained with different cross-linking ratio (viz. 1:2, 1:4 and 1:8 on molar basis with the cross-linker) to protect the lactone ring from hydrolysis and to prolong the release kinetics of CAM. Crystalline (F 1:2 , F 1:4 and F 1:8 ) and paracrystalline NS formulations were prepared. XRPD, DSC and FTIR studies confirmed the interactions of CAM with NS. XRPD showed that the crystallinity of CAM decreased after loading. CAM was loaded as much as 21%, 37% and 13% w/w in F 1:2 , F 1:4 and F 1:8 , respectively while the paracrystalline NS formulations gave a loading of about 10% w/w or lower. The particle sizes of the loaded NS formulations were between 450 and 600 nm with low polydispersity indices. The zeta potentials were sufficiently high (À20 to À25 mV) to obtain a stable colloidal nanosuspension. The in vitro studies indicated a slow and prolonged CAM release over a period of 24 h. The NS formulations protected the lactone ring of CAM after their incubation in physiological conditions at 37°C for 24 h with a 80% w/w of intact lactone ring when compared to only around 20% w/w of plain CAM. The cytotoxicity studies on HT-29 cells showed that the CAM formulations were more cytotoxic than plain CAM after 24 h of incubation.

1994/1997–2004/2007: changes in the requests to the help-line of the Institute of Clinical Sexology

Objective: To describe changes in the requests presented by men and women calling the help-line o... more

Lactadherin-derived peptides as antiviral agents

Filoviruses Utilize Glycosaminoglycans for Their Attachment to Target Cells

Journal of Virology, 2013

Filoviruses are the cause of severe hemorrhagic fever in human and nonhuman primates. The envelop... more Filoviruses are the cause of severe hemorrhagic fever in human and nonhuman primates. The envelope glycoprotein (GP), responsible for both receptor binding and fusion of the virus envelope with the host cell membrane, has been demonstrated to interact with multiple molecules in order to enhance entry into host cells. Here we have demonstrated that filoviruses utilize glycosaminoglycans, and more specifically heparan sulfate proteoglycans, for their attachment to host cells. This interaction is mediated by GP and does not require the presence of the mucin domain. Both the degree of sulfation and the structure of the carbohydrate backbone play a role in the interaction with filovirus GPs. This new step of filovirus interaction with host cells can potentially be a new target for antiviral strategies. As such, we were able to inhibit filovirus GP-mediated infection using carrageenan, a broad-spectrum microbicide that mimics heparin, and also using the antiviral dendrimeric peptide SB105-A10, which interacts with heparan sulfate, antagonizing the binding of the virus to cells.

Modulation of HIV-LTR activity by ras oncogenes

The New Microbiologica: official journal of the Italian Society for Medical Virology (SIVIM)

Cotransfection of NIH 3T3 cells with a mammalian expression vector containing a v-Ha-ras gene, to... more Cotransfection of NIH 3T3 cells with a mammalian expression vector containing a v-Ha-ras gene, together with a plasmid carrying the human immunodeficiency virus (HIV) long terminal repeat (LTR) linked to the chloramphenicol acetyl transferase (CAT) reporter gene, significantly stimulated CAT activity. High HIV LTR activation was also observed in cell lines carrying stably transfected ras oncogenes, activated by point mutation or amplification. By contrast an inactivated form of ras (Ha-ras Asn-17) did not stimulate the HIV-LTR but strongly inhibited its basal activity. Activation of the p21ras protein may thus be one of the signals that regulate LTR driven transcription during HIV infection.

The influence of caregiver burden on sexual intimacy and marital satisfaction in couples with an Alzheimer spouse

International Journal of Clinical Practice, 2007

Reduction of spontaneous autoimmune diabetes-prone BB rats with a novel immunosuppressant fusidic acid. Effect on T-cell proliferation and production of interferon-gamma

Immunology

Diabetes-prone (DP) BB rats spontaneously develop a hyperglycaemic condition which closely resemb... more Diabetes-prone (DP) BB rats spontaneously develop a hyperglycaemic condition which closely resembles human insulin-dependent diabetes mellitus (IDDM), both in terms of clinical and histological features. The incidence of IDDM was significantly reduced when these animals were treated with 2 or 4 mg fusidic acid (FA)/day i.m. from day 30 to day 120 of age. In addition, the mean insulitis score was significantly diminished in the animals treated with FA compared to both vehicle-treated and untreated controls. Finally, 2 mg/day of FA i.m. prevented cell proliferation and interferon-gamma secretion from peripheral blood mononuclear cells upon ex vivo stimulation with concanavalin A. The capacity of FA to substantially reduce the incidence of autoimmune diabetes in a well-known animal model of human IDDM supports previous observations regarding the immunosuppressive properties of FA and its potential use in the treatment of human autoimmune diabetes.

[Early diagnosis of HCMV infections in patients undergoing transplantation of the kidney]

Giornale di batteriologia, virologia ed immunologia

HCMV infection is a major cause of morbidity and mortality following kidney transplantation. Clin... more HCMV infection is a major cause of morbidity and mortality following kidney transplantation. Clinical diagnosis is difficult, and rapid and sensitive diagnostic methods are needed since antiviral therapy is available. One hundred-forty-five consecutive kidney-transplanted patients were studied during a period of three months after transplantation. For laboratory diagnosis of HCMV infection, we looked for the presence of pp-65 antigen in polymorphonuclear leukocytes, HCMV-DNA and IgM. Demonstration of HCMV pp-65 antigen by immunofluorescence and HCMV DNA by PCR in leukocytes were efficient methods for early diagnosis of infection.

Murine cytomegalovirus induces expression and enzyme activity of cellular dihydrofolate reductase in quiescent cells

The Journal of general virology, 1998

Murine cytomegalovirus (MCMV) productively infects quiescent fibroblasts in which the levels of n... more Murine cytomegalovirus (MCMV) productively infects quiescent fibroblasts in which the levels of nucleoside triphosphate precursors and cell functions involved in DNA metabolism are minimal. It appears that MCMV has evolved molecular pathways in order to ensure the presence of nucleoside triphosphate precursors for the viral DNA polymerase. Here, we report that MCMV infection of quiescent NIH 3T3 cells markedly stimulates transcription, expression and activity of the cellular dihydrofolate reductase (DHFR), a key enzyme in the synthesis of DNA precursors. DHFR stimulation by MCMV is sensitive to UV irradiation and seems to depend on expression of the viral immediate-early protein pp89. Finally, it has been demonstrated that suppression of virus-induced DHFR activity by the specific inhibitor methotrexate prevents MCMV DNA replication. These observations indicate that induction of host cell DHFR activity by MCMV is required for viral DNA synthesis in quiescent fibroblasts.

Inhibition of proinflammatory and innate immune signaling pathways by a cytomegalovirus RIP1-interacting protein

Proceedings of the National Academy of Sciences of the United States of America, Jan 26, 2008

TNFalpha is an important cytokine in antimicrobial immunity and inflammation. The receptor-intera... more TNFalpha is an important cytokine in antimicrobial immunity and inflammation. The receptor-interacting protein RIP1 is an essential component of the TNF receptor 1 signaling pathway that mediates the activation of NF-kappaB, MAPKs, and programmed cell death. It also transduces signals derived from Toll-like receptors and intracellular sensors of DNA damage and double-stranded RNA. Here, we show that the murine CMV M45 protein binds to RIP1 and inhibits TNFalpha-induced activation of NF-kappaB, p38 MAPK, and caspase-independent cell death. M45 also inhibited NF-kappaB activation upon stimulation of Toll-like receptor 3 and ubiquitination of RIP1, which is required for NF-kappaB activation. Hence, M45 functions as a viral inhibitor of RIP1-mediated signaling. The results presented here reveal a mechanism of viral immune subversion and demonstrate how a viral protein can simultaneously block proinflammatory and innate immune signaling pathways by interacting with a central mediator mol...

Highly sulfated K5 Escherichia coli polysaccharide derivatives inhibit respiratory syncytial virus infectivity in cell lines and human tracheal-bronchial histocultures

by Marco Volante, Marco Rusnati, David Lembo, and Valeria Cagno

Antimicrobial agents and chemotherapy, 2014

Respiratory syncytial virus (RSV) exploits cell surface heparan sulfate proteoglycans (HSPGs) as ... more Respiratory syncytial virus (RSV) exploits cell surface heparan sulfate proteoglycans (HSPGs) as attachment receptors. The interaction between RSV and HSPGs thus presents an attractive target for the development of novel inhibitors of RSV infection. In this study, selective chemical modification of the Escherichia coli K5 capsular polysaccharide was used to generate a collection of sulfated K5 derivatives with a backbone structure that mimics the heparin/heparan sulfate biosynthetic precursor. The screening of a series of N-sulfated (K5-NS), O-sulfated (K5-OS), and N,O-sulfated (K5-N,OS) derivatives with different degrees of sulfation revealed the highly sulfated K5 derivatives K5-N,OS(H) and K5-OS(H) to be inhibitors of RSV. Their 50% inhibitory concentrations were between 1.07 nM and 3.81 nM in two different cell lines, and no evidence of cytotoxicity was observed. Inhibition of RSV infection was maintained in binding and attachment assays but not in preattachment assays. Moreover...

Prions: a mystery unravelled?

Rivista di biologia

Prions result in fatal degeneration of the central nervous system (CNS) in the form of diseases k... more Prions result in fatal degeneration of the central nervous system (CNS) in the form of diseases known as transmissible spongiform encephalopathies (TSEs). The discovery in 1996 of a new variant of Creutzfeldt-Jakob disease (a human TSE) and experimental confirmation that it is caused by the prion strain responsible for bovine spongiform encephalopathy (BSE) has greatly spurred research in this field. The mechanism underlying prion propagation is now reasonably clear. Prions multiply, in fact, by stimulating their hosts to produce proteins that are initially normal, but acquire an abnormal, prion-like conformation during the coiling stage. A fuller understanding of this mechanism could lead to the employment of molecules capable of making prion proteins revert to the normal conformation in the treatment of both TSEs and other serious CNS disorders.

Identification of Equine Lactadherin-Derived Peptides that Inhibit Rotavirus Infection via Integrin Receptor Competition

by David Lembo, Barbara Coulson, and Amedeo Conti

The Journal of biological chemistry, Jan 26, 2015

Human rotavirus is the leading cause of severe gastroenteritis in infants and children under the ... more Human rotavirus is the leading cause of severe gastroenteritis in infants and children under the age of five years in both developed and developing countries. Human lactadherin, a milk-fat globule membrane (MFGM) glycoprotein, inhibits human rotavirus infection in vitro, whereas bovine lactadherin is not active. Moreover, it protects breastfed infants against symptomatic rotavirus infections. To explore the potential antiviral activity of lactadherin sourced by equines we undertook a proteomic analysis of MFGM proteins from donkey milk and elucidated its amino acid sequence. Alignment of the human, bovine and donkey lactadherin sequences revealed the presence of an Asp-Gly-Glu (DGE) α2β1 integrin-binding motif in the N-terminal domain of donkey sequence only. Since integrin α2β1 plays a critical role during early steps of rotavirus host cell adhesion, we tested a mini-library of donkey lactadherin-derived peptides containing DGE sequence for anti-rotavirus activity. A 20 amino acid ...

Early inhibitors of human cytomegalovirus: State-of-art and therapeutic perspectives

by Beatrice Mercorelli and David Lembo

Pharmacology & Therapeutics, 2011

Human cytomegalovirus (HCMV) infection is associated with severe morbidity and mortality in immun... more Human cytomegalovirus (HCMV) infection is associated with severe morbidity and mortality in immunocompromised individuals, mainly transplant recipients and AIDS patients, and is the most frequent cause of congenital malformations in newborn children. To date, few drugs are licensed for the treatment of HCMV infections, most of which target the viral DNA polymerase and suffer from many drawbacks, including long-term toxicity, low potency, and poor bioavailability. In addition, the emergence of drug-resistant viral strains is becoming an increasing problem for disease management. Finally, none of the current anti-HCMV drugs have been approved for the treatment of congenital infections. For all these reasons, there is still a strong need for new anti-HCMV drugs with novel mechanisms of action. The first events of the virus replication cycle, including attachment, entry, immediate-early gene expression, and immediate-early functions-in particular that of Immediate-Early 2 protein-represent attractive targets for the development of novel antiviral compounds. Such inhibitors would block not only the expression of viral immediate-early proteins, which play a key role in the pathogenesis of HCMV infection, but also the host immunomodulation and the changes to cell physiology induced by the first events of virus infection. This review describes the current knowledge on the initial phases of HCMV replication, their validation as potential novel antiviral targets, and the development of compounds that block such processes.

Hexagonal-shaped chondroitin sulfate self-assemblies have exalted anti-HSV-2 activity

Carbohydrate Polymers, 2015

The initial step in mucosal infection by the herpes simplex virus type 2 (HSV-2) requires its bin... more The initial step in mucosal infection by the herpes simplex virus type 2 (HSV-2) requires its binding to certain glycosaminoglycans naturally present on host cell membranes. We took advantage of this interaction to design biomimetic supramolecular hexagonal-shaped nanoassemblies composed of chondroitin sulfate having exalted anti-HSV-2 activity in comparison with native chondroitin sulfate. Nanoassemblies were formed by mixing hydrophobically-modified chondroitin sulfate with α-cyclodextrin in water. Optimization of alkyl chain length grafted on chondroitin sulfate and the ratio between hydrophobically-modified chondroitin sulfate and α-cyclodextrin showed that more cohesive and well-structured nanoassemblies were obtained using higher α-cyclodextrin concentration and longer alkyl chain lengths. A structure-activity relationship was found between anti-HSV-2 activity and the amphiphilic nature of hydrophobically-modified chondroitin sulfate. Also, antiviral activity of hexagonal nanoassemblies against HSV-2 was further improved in comparison with hydrophobically-modified chondroitin sulfate. This work suggests a new biomimetic formulation approach that can be extended to other heparan-sulfate-dependent viruses.

Cytomegalovirus stimulates the expression of cellular enzymes involved in nucleotides biosynthesis

Journal of Clinical Virology, 1999

The AGMA1 poly(amidoamine) inhibits the infectivity of herpes simplex virus in cell lines, in human cervicovaginal histocultures, and in vaginally infected mice

Biomaterials, 2016

The development of topical microbicides is a valid approach to protect the genital mucosa from se... more The development of topical microbicides is a valid approach to protect the genital mucosa from sexually transmitted infections that cannot be contained with effective vaccination, like HSV and HIV infections. A suitable target of microbicides is the interaction between viral proteins and cell surface heparan sulfate proteoglycans (HSPGs). AGMA1 is a prevailingly cationic agmatine-containing polyamidoamine polymer previously shown to inhibit HSPGs dependent viruses, including HSV-1, HSV-2, and HPV-16. The aim of this study was to elucidate the mechanism of action of AGMA1 against HSV infection and assess its antiviral efficacy and biocompatibility in preclinical models. The results show AGMA1 to be a non-toxic inhibitor of HSV infectivity in cell cultures and human cervicovaginal histocultures. Moreover, it significantly reduced the burden of infection of HSV-2 genital infection in mice. The investigation of the mechanism of action revealed that AGMA1 reduces cells susceptibility to virus infection by binding to cell surface HSPGs thereby preventing HSV attachment. This study indicates that AGMA1 is a promising candidate for the development of a topical microbicide to prevent sexually transmitted HSV infections.

Novel Antipathogenic Peptides

The human cytomegalovirus

Pharmacology Therapeutics, Jun 1, 2003

Human cytomegalovirus (HCMV), a betaherpesvirus, represents the major infectious cause of birth d... more Human cytomegalovirus (HCMV), a betaherpesvirus, represents the major infectious cause of birth defects, as well as an important pathogen for immunocompromised individuals. The viral nucleocapsid containing a linear double-stranded DNA of 230 kb is surrounded by a proteinaceous tegument, which is itself enclosed by a loosely applied lipid bilayer. Expression of the HCMV genome is controlled by a cascade of transcriptional events that leads to the synthesis of three categories of viral proteins designated as immediate-early, early, and late. Clinical manifestations can be seen following primary infection, reinfection, or reactivation. About 10% of infants are infected by the age of 6 months following transmission from their mothers via the placenta, during delivery, or by breastfeeding. HCMV is a significant post-allograft pathogen and contributes to graft loss independently from graft rejection. Histopathologic examination of necropsy tissues demonstrates that the virus enters via the epithelium of the upper alimentary, respiratory, or genitourinary tracts. Hematogenous spreading is typically followed by infection of ductal epithelial cells. Infections are kept under control by the immune system. However, total HCMV clearance is rarely achieved, and the viral genome remains at selected sites in a latent state. Virological and molecular detection of HCMV, as well as serological demonstration of a specific immune response, are used for diagnosis. Treatment of HCMV infections is difficult because there are few options. The presently available drugs produced a significant clinical improvement, but suffer from poor oral bioavailability, low potency, development of resistance in clinical practice, and dose-limiting toxicities. D

The L1 protein of human papilloma virus 16 expressed by a fowlpox virus recombinant can assemble into virus-like particles in mammalian cell lines but elicits a non-neutralising humoral response

Antiviral Research, 2015

Human papilloma virus (HPV)-16 is the prevalent genotype associated with cervical tumours. Virus-... more Human papilloma virus (HPV)-16 is the prevalent genotype associated with cervical tumours. Virus-likeparticle (VLP)-based vaccines have proven to be effective in limiting new infections of high-risk HPVs, but their high cost has hampered their use, especially in the poor developing countries. Avipox-based recombinants are replication-restricted to avian species and represent efficient and safe vectors also for immunocompromised hosts, as they can elicit a complete immune response. A new fowlpox virus recombinant encoding HPV-L1 (FP L1 ) was engineered and evaluated side-by-side with a FP recombinant co-expressing L1 and green fluorescent protein (FP L1GFP ) for correct expression of L1 in vitro in different cell lines, as confirmed by Western blotting, immunofluorescence, real-time PCR, and electron microscopy. Mice were also immunised to determine its immunogenicity. Here, we demonstrate that the FP L1 recombinant better expresses L1 in the absence of GFP, correctly assembles structured capsomers into VLPs, and elicits an immune response in a preclinical animal model. To our knowledge, this is the first report of HPV VLPs assembled in eukaryotic cells using an avipox recombinant.

Cyclodextrin based nanosponges encapsulating camptothecin

Camptothecin (CAM), a plant alkaloid and a potent antitumor agent, has a limited therapeutic util... more Camptothecin (CAM), a plant alkaloid and a potent antitumor agent, has a limited therapeutic utility because of its poor aqueous solubility, lactone ring instability and serious side effects. Cyclodextrin-based nanosponges (NS) are a novel class of cross-linked derivatives of cyclodextrins. They have been used to increase the solubility of poorly soluble actives, to protect the labile groups and control the release. This study aimed at formulating complexes of CAM with three types of b-cyclodextrin NS obtained with different cross-linking ratio (viz. 1:2, 1:4 and 1:8 on molar basis with the cross-linker) to protect the lactone ring from hydrolysis and to prolong the release kinetics of CAM. Crystalline (F 1:2 , F 1:4 and F 1:8 ) and paracrystalline NS formulations were prepared. XRPD, DSC and FTIR studies confirmed the interactions of CAM with NS. XRPD showed that the crystallinity of CAM decreased after loading. CAM was loaded as much as 21%, 37% and 13% w/w in F 1:2 , F 1:4 and F 1:8 , respectively while the paracrystalline NS formulations gave a loading of about 10% w/w or lower. The particle sizes of the loaded NS formulations were between 450 and 600 nm with low polydispersity indices. The zeta potentials were sufficiently high (À20 to À25 mV) to obtain a stable colloidal nanosuspension. The in vitro studies indicated a slow and prolonged CAM release over a period of 24 h. The NS formulations protected the lactone ring of CAM after their incubation in physiological conditions at 37°C for 24 h with a 80% w/w of intact lactone ring when compared to only around 20% w/w of plain CAM. The cytotoxicity studies on HT-29 cells showed that the CAM formulations were more cytotoxic than plain CAM after 24 h of incubation.

1994/1997–2004/2007: changes in the requests to the help-line of the Institute of Clinical Sexology

Objective: To describe changes in the requests presented by men and women calling the help-line o... more

Lactadherin-derived peptides as antiviral agents

Filoviruses Utilize Glycosaminoglycans for Their Attachment to Target Cells

Journal of Virology, 2013

Filoviruses are the cause of severe hemorrhagic fever in human and nonhuman primates. The envelop... more Filoviruses are the cause of severe hemorrhagic fever in human and nonhuman primates. The envelope glycoprotein (GP), responsible for both receptor binding and fusion of the virus envelope with the host cell membrane, has been demonstrated to interact with multiple molecules in order to enhance entry into host cells. Here we have demonstrated that filoviruses utilize glycosaminoglycans, and more specifically heparan sulfate proteoglycans, for their attachment to host cells. This interaction is mediated by GP and does not require the presence of the mucin domain. Both the degree of sulfation and the structure of the carbohydrate backbone play a role in the interaction with filovirus GPs. This new step of filovirus interaction with host cells can potentially be a new target for antiviral strategies. As such, we were able to inhibit filovirus GP-mediated infection using carrageenan, a broad-spectrum microbicide that mimics heparin, and also using the antiviral dendrimeric peptide SB105-A10, which interacts with heparan sulfate, antagonizing the binding of the virus to cells.

Modulation of HIV-LTR activity by ras oncogenes

The New Microbiologica: official journal of the Italian Society for Medical Virology (SIVIM)

Cotransfection of NIH 3T3 cells with a mammalian expression vector containing a v-Ha-ras gene, to... more Cotransfection of NIH 3T3 cells with a mammalian expression vector containing a v-Ha-ras gene, together with a plasmid carrying the human immunodeficiency virus (HIV) long terminal repeat (LTR) linked to the chloramphenicol acetyl transferase (CAT) reporter gene, significantly stimulated CAT activity. High HIV LTR activation was also observed in cell lines carrying stably transfected ras oncogenes, activated by point mutation or amplification. By contrast an inactivated form of ras (Ha-ras Asn-17) did not stimulate the HIV-LTR but strongly inhibited its basal activity. Activation of the p21ras protein may thus be one of the signals that regulate LTR driven transcription during HIV infection.

The influence of caregiver burden on sexual intimacy and marital satisfaction in couples with an Alzheimer spouse

International Journal of Clinical Practice, 2007

Reduction of spontaneous autoimmune diabetes-prone BB rats with a novel immunosuppressant fusidic acid. Effect on T-cell proliferation and production of interferon-gamma

Immunology

Diabetes-prone (DP) BB rats spontaneously develop a hyperglycaemic condition which closely resemb... more Diabetes-prone (DP) BB rats spontaneously develop a hyperglycaemic condition which closely resembles human insulin-dependent diabetes mellitus (IDDM), both in terms of clinical and histological features. The incidence of IDDM was significantly reduced when these animals were treated with 2 or 4 mg fusidic acid (FA)/day i.m. from day 30 to day 120 of age. In addition, the mean insulitis score was significantly diminished in the animals treated with FA compared to both vehicle-treated and untreated controls. Finally, 2 mg/day of FA i.m. prevented cell proliferation and interferon-gamma secretion from peripheral blood mononuclear cells upon ex vivo stimulation with concanavalin A. The capacity of FA to substantially reduce the incidence of autoimmune diabetes in a well-known animal model of human IDDM supports previous observations regarding the immunosuppressive properties of FA and its potential use in the treatment of human autoimmune diabetes.

[Early diagnosis of HCMV infections in patients undergoing transplantation of the kidney]

Giornale di batteriologia, virologia ed immunologia

HCMV infection is a major cause of morbidity and mortality following kidney transplantation. Clin... more HCMV infection is a major cause of morbidity and mortality following kidney transplantation. Clinical diagnosis is difficult, and rapid and sensitive diagnostic methods are needed since antiviral therapy is available. One hundred-forty-five consecutive kidney-transplanted patients were studied during a period of three months after transplantation. For laboratory diagnosis of HCMV infection, we looked for the presence of pp-65 antigen in polymorphonuclear leukocytes, HCMV-DNA and IgM. Demonstration of HCMV pp-65 antigen by immunofluorescence and HCMV DNA by PCR in leukocytes were efficient methods for early diagnosis of infection.

Murine cytomegalovirus induces expression and enzyme activity of cellular dihydrofolate reductase in quiescent cells

The Journal of general virology, 1998

Murine cytomegalovirus (MCMV) productively infects quiescent fibroblasts in which the levels of n... more Murine cytomegalovirus (MCMV) productively infects quiescent fibroblasts in which the levels of nucleoside triphosphate precursors and cell functions involved in DNA metabolism are minimal. It appears that MCMV has evolved molecular pathways in order to ensure the presence of nucleoside triphosphate precursors for the viral DNA polymerase. Here, we report that MCMV infection of quiescent NIH 3T3 cells markedly stimulates transcription, expression and activity of the cellular dihydrofolate reductase (DHFR), a key enzyme in the synthesis of DNA precursors. DHFR stimulation by MCMV is sensitive to UV irradiation and seems to depend on expression of the viral immediate-early protein pp89. Finally, it has been demonstrated that suppression of virus-induced DHFR activity by the specific inhibitor methotrexate prevents MCMV DNA replication. These observations indicate that induction of host cell DHFR activity by MCMV is required for viral DNA synthesis in quiescent fibroblasts.

Inhibition of proinflammatory and innate immune signaling pathways by a cytomegalovirus RIP1-interacting protein

Proceedings of the National Academy of Sciences of the United States of America, Jan 26, 2008

TNFalpha is an important cytokine in antimicrobial immunity and inflammation. The receptor-intera... more TNFalpha is an important cytokine in antimicrobial immunity and inflammation. The receptor-interacting protein RIP1 is an essential component of the TNF receptor 1 signaling pathway that mediates the activation of NF-kappaB, MAPKs, and programmed cell death. It also transduces signals derived from Toll-like receptors and intracellular sensors of DNA damage and double-stranded RNA. Here, we show that the murine CMV M45 protein binds to RIP1 and inhibits TNFalpha-induced activation of NF-kappaB, p38 MAPK, and caspase-independent cell death. M45 also inhibited NF-kappaB activation upon stimulation of Toll-like receptor 3 and ubiquitination of RIP1, which is required for NF-kappaB activation. Hence, M45 functions as a viral inhibitor of RIP1-mediated signaling. The results presented here reveal a mechanism of viral immune subversion and demonstrate how a viral protein can simultaneously block proinflammatory and innate immune signaling pathways by interacting with a central mediator mol...

Highly sulfated K5 Escherichia coli polysaccharide derivatives inhibit respiratory syncytial virus infectivity in cell lines and human tracheal-bronchial histocultures

by Marco Volante, Marco Rusnati, David Lembo, and Valeria Cagno

Antimicrobial agents and chemotherapy, 2014

Respiratory syncytial virus (RSV) exploits cell surface heparan sulfate proteoglycans (HSPGs) as ... more Respiratory syncytial virus (RSV) exploits cell surface heparan sulfate proteoglycans (HSPGs) as attachment receptors. The interaction between RSV and HSPGs thus presents an attractive target for the development of novel inhibitors of RSV infection. In this study, selective chemical modification of the Escherichia coli K5 capsular polysaccharide was used to generate a collection of sulfated K5 derivatives with a backbone structure that mimics the heparin/heparan sulfate biosynthetic precursor. The screening of a series of N-sulfated (K5-NS), O-sulfated (K5-OS), and N,O-sulfated (K5-N,OS) derivatives with different degrees of sulfation revealed the highly sulfated K5 derivatives K5-N,OS(H) and K5-OS(H) to be inhibitors of RSV. Their 50% inhibitory concentrations were between 1.07 nM and 3.81 nM in two different cell lines, and no evidence of cytotoxicity was observed. Inhibition of RSV infection was maintained in binding and attachment assays but not in preattachment assays. Moreover...

Prions: a mystery unravelled?

Rivista di biologia

Prions result in fatal degeneration of the central nervous system (CNS) in the form of diseases k... more Prions result in fatal degeneration of the central nervous system (CNS) in the form of diseases known as transmissible spongiform encephalopathies (TSEs). The discovery in 1996 of a new variant of Creutzfeldt-Jakob disease (a human TSE) and experimental confirmation that it is caused by the prion strain responsible for bovine spongiform encephalopathy (BSE) has greatly spurred research in this field. The mechanism underlying prion propagation is now reasonably clear. Prions multiply, in fact, by stimulating their hosts to produce proteins that are initially normal, but acquire an abnormal, prion-like conformation during the coiling stage. A fuller understanding of this mechanism could lead to the employment of molecules capable of making prion proteins revert to the normal conformation in the treatment of both TSEs and other serious CNS disorders.