Publications by authors named "Ilaria Schiavoni"

19 Publications

  • Page 1 of 1

Increased T-helper Cell 2 Response in Infants With Respiratory Syncytial Virus Bronchiolitis Hospitalized Outside Epidemic Peak.

Pediatr Infect Dis J 2020 01;39(1):61-67

From the Department of Pediatrics, Sapienza University of Rome, Rome, Italy.

Aim: To test the hypothesis that the balance of type-1/type-2 immune response differs between infants hospitalized with respiratory syncytial virus (RSV) bronchiolitis during the peak months and those during the nonpeak months.

Methods: We prospectively enrolled 90 unrelated full-term previously healthy infants hospitalized during the first year of life for RSV sole bronchiolitis over 2 epidemics (November 2016 to April 2017 and October 2017 to April 2018). We stratified infants as follows: hospitalized during the peak months (n: 71) and during the nonpeak months (n: 19). The frequencies of CD4+ producing interferon (IFN)-γ and interleukin (IL)-4 and of CD8+ producing IFN-γ T cells were measured by flow cytometry from infant peripheral whole blood. The T-helper cell (Th2) polarization index was calculated as the ratio between CD4+ T cells producing IL-4 and CD4+ T cells producing IFN-γ.

Results: Infants hospitalized during nonpeak months were significantly less frequently breast-fed, had a higher eosinophils count, a significantly higher percentage of CD4+ T cells producing IL-4 and higher Th2 polarization index than infants hospitalized during the peak months.

Conclusions: We elucidated the presence of different endotypes in infants with RSV sole bronchiolitis. Previously healthy full-term infants hospitalized during the nonpeak months seem to be more likely those with a possible predisposition to atopy.
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http://dx.doi.org/10.1097/INF.0000000000002505DOI Listing
January 2020

Diagnostic performance of commercial serological assays measuring Bordetella pertussis IgG antibodies.

Diagn Microbiol Infect Dis 2018 Mar 13;90(3):157-162. Epub 2017 Nov 13.

Department of Infectious Diseases, Istituto Superiore di Sanità, Rome, Italy. Electronic address:

Due to their specificity to B. pertussis antigens, immunoglobulin G (IgG) antibodies should be measured primarily for diagnosing pertussis. We compared the diagnostic performance of commercially available enzyme-linked immunosorbent assays (ELISAs) and chemiluminescent immunoassays (CLIAs) measuring IgG to B. pertussis antigens. An in-house ELISA with purified pertussis toxin (PT) was used as reference system. Commercial assays using PT only as coating antigen showed better performance as compared to those using a mixture of different antigens. The best diagnostic performances were achieved by CLIAs. Results were analyzed using a dual cutoff of either ≥125IU/mL anti-PT IgG or ≥62IU/mL anti-PT IgG for the in-house ELISA and accordingly to package inserts for commercial assays. Using the in-house ELISA at a 62 IU/mL cutoff, as the gold standard for interpretation of results from the commercial kits, resulted in lower sensitivity and higher specificity as compared to 125IU/mL, thus, it may be especially useful in outbreak situations when high specificity is required.
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http://dx.doi.org/10.1016/j.diagmicrobio.2017.11.006DOI Listing
March 2018

CD38 modulates respiratory syncytial virus-driven proinflammatory processes in human monocyte-derived dendritic cells.

Immunology 2018 05 18;154(1):122-131. Epub 2017 Dec 18.

Department of Infectious Diseases, Istituto Superiore di Sanità, Rome, Italy.

Respiratory syncytial virus (RSV) is the most common cause of hospitalization due to bronchiolitis in infants. Although the mechanisms behind this association are not completely elucidated, they appear to involve an excessive immune response causing lung pathology. Understanding the host response to RSV infection may help in the identification of targets for therapeutic intervention. We infected in-vitro human monocyte-derived dendritic cells (DCs) with RSV and analysed various aspects of the cellular response. We found that RSV induces in DCs the expression of CD38, an ectoenzyme that catalyses the synthesis of cyclic ADPR (cADPR). Remarkably, CD38 was under the transcriptional control of RSV-induced type I interferon (IFN). CD38 and a set of IFN-stimulated genes (ISGs) were inhibited by the anti-oxidant N-acetyl cysteine. When CD38-generated cADPR was restrained by 8-Br-cADPR or kuromanin, a flavonoid known to inhibit CD38 enzymatic activity, RSV-induced type I/III IFNs and ISGs were markedly reduced. Taken together, these results suggest a key role of CD38 in the regulation of anti-viral responses. Inhibition of CD38 enzymatic activity may represent an encouraging approach to reduce RSV-induced hyperinflammation and a novel therapeutic option to treat bronchiolitis.
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http://dx.doi.org/10.1111/imm.12873DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5904717PMC
May 2018

Invasion of Dendritic Cells, Macrophages and Neutrophils by the Bordetella Adenylate Cyclase Toxin: A Subversive Move to Fool Host Immunity.

Toxins (Basel) 2017 09 21;9(10). Epub 2017 Sep 21.

Institute of Microbiology of the CAS, v. v. i., Prague 142 20, Czech Republic.

Adenylate cyclase toxin (CyaA) is released in the course of infection in the host's respiratory tract in order to suppress its early innate and subsequent adaptive immune defense. CD11b-expressing dendritic cells (DC), macrophages and neutrophils are professional phagocytes and key players of the innate immune system that provide a first line of defense against invading pathogens. Recent findings revealed the capacity of CyaA to intoxicate DC with high concentrations of 3',5'-cyclic adenosine monophosphate (cAMP), which ultimately skews the host immune response towards the expansion of Th17 cells and regulatory T cells. CyaA-induced cAMP signaling swiftly incapacitates opsonophagocytosis, oxidative burst and NO-mediated killing of bacteria by neutrophils and macrophages. The subversion of host immune responses by CyaA after delivery into DC, macrophages and neutrophils is the subject of this review.
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http://dx.doi.org/10.3390/toxins9100293DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5666340PMC
September 2017

Antibody mimicry, receptors and clinical applications.

Hum Antibodies 2017 ;25(3-4):75-85

Laboratory of Immunogenetics, Department of Medical Sciences, University of Torino, Torino 10126, Italy.

This review focuses on the concept of antibodies acting as receptor agonists and antagonists, and on the potential relevance of this notion in applied medicine. Antibodies are composed of three functional units: two antigen-binding fragments (Fabs) that confer antigen specificity and one constant fragment (Fc) linking antibodies to immune effector functions. The proof-of-concept that large amounts of highly specific and homogeneous antibodies could be produced was provided in 1975 by César Milstein and Georges Köhler. These monoclonal antibody (mAb) reagents started a revolution in medical research, diagnostics, and clinical applications. Alongside diagnostic applications, mAbs were successfully used in vivo: (i) to bind (neutralize/antagonize) antigens expressed on the surface of tumor cells; (ii) to activate immune effector mechanisms; (iii) to crosslink plasma membrane receptors and hence activate therapeutic signaling pathways; and lastly, (iv) the technique was expanded to produce bispecific mAbs, which can bind two different antigens while retaining the ability to activate immune effector functions. The abilities of mAbs to bind, transduce signals, and exert immunostimulatory agonistic capacities are the central issues of this review. The starting point is that some mAbs operate as molecular agonists, substituting for the natural ligand of the receptor. Our analysis is restricted to mAbs that act as receptor agonist/antagonists by either mimicking ligand binding, or through allosteric modulation mediated by binding sites that are topographically distinct from the orthosteric binding site. Functional considerations based on the agonistic stimulation of human CD38 by specific mAbs as surrogate ligands are described as examples of the features of such molecules.
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http://dx.doi.org/10.3233/HAB-160305DOI Listing
February 2018

Persistence of T-cell immune response induced by two acellular pertussis vaccines in children five years after primary vaccination.

New Microbiol 2016 Jan;39(1):35-47

Anti-Infectious Immunity Unit, Department of Infectious, Parasitic and immune-mediated Diseases, Istituto Superiore di Sanità, Rome, Italy.

The resurgence of pertussis suggests the need for greater efforts to understand the long-lasting protective responses induced by vaccination. In this paper we dissect the persistence of T memory responses induced by primary vaccination with two different acellular pertussis (aP) vaccines, hexavalent Hexavac® vaccine (Hexavac) (Sanofi Pasteur MSD) and Infanrix hexa® (Infanrix) (Glaxo-SmithKline Biologicals). We evaluated magnitude and duration of T-cell responses to pertussis toxin (PT) by measuring T-cell proliferation, cytokines (IL-2 and IFNγ) production and memory subsets in two groups of children 5 years after primary vaccination. Some of the enrolled children received only primary vaccination, while others had the pre-school boost dose. Positive T-cell responses to PT were detected in 36% of children. Percentage of responsive children, T-cell proliferation and CD4IL-2+ cells were significantly higher in the children primed with Hexavac than in those who received Infanrix vaccine. No major effects of the boost on PT-specific proliferation were observed. Overall, our data documented a persistence of T-cell memory against PT in a minor fraction of children 5 years after primary vaccination. The different responses induced by Hexavac and Infanrix vaccine could rely on differences in PT inactivation process or excipients/adjuvants formulations.
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January 2016

Unconventional, adenosine-producing suppressor T cells induced by dendritic cells exposed to BPZE1 pertussis vaccine.

J Leukoc Biol 2015 Oct 18;98(4):631-9. Epub 2015 Jun 18.

*Anti-Infectious Immunity Unit, Department of Infectious Parasitic and Immune-Mediated Diseases, Istituto Superiore di Sanità, Rome, Italy; INSERM U1019, Lille, France; CNRS UMR8204, Lille, France; Institut Pasteur de Lille, Center for Infection and Immunity, Lille, France; University of Lille Nord de France, Lille, France; Laboratory of Immunogenetics, Department of Medical Sciences and CERMS, University of Torino, Torino, Italy; **Transplantation Immunology Service, Città della Salute e della Scienza Hospital, Torino, Italy.

BPZE1 is a live attenuated pertussis vaccine that successfully completed a phase 1 safety trial. This article describes the induction of unconventional suppressor T cells-producing ADO by MDDCs exposed to BPZE1 (BPZE1-DC) through distinct ectoenzymatic pathways that limit the damaging effect of inflammation. BPZE1-DC induces CD4(+) and CD8(+) T lymphocytes to express 2 sets of ectoenzymes generating ADO: 1 set is part of the conventional CD39/CD73 pathway, which uses ATP as substrate, whereas the other is part of the CD38/CD203a/CD73 pathway and metabolizes NAD(+). The contribution of the ADO-generating ectoenzymes in the regulatory response was shown by: 1) selective inhibition of the enzymatic activities of CD39, CD73, and CD38; 2) the ability of suppressor T cells to convert exogenously added ATP and NAD(+) to ADO; and 3) a positive correlation between ectoenzyme expression, ADO levels, and suppression abilities. Thus, T lymphocytes activated by BPZE1-DC shift to a suppressor stage, through the expression of ectoenzyme networks, and are able to convert extracellular nucleotides into ADO, which may explain the potent anti-inflammatory properties of BPZE1 observed in several murine models.
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http://dx.doi.org/10.1189/jlb.3A0315-101RDOI Listing
October 2015

Live attenuated B. pertussis BPZE1 rescues the immune functions of Respiratory Syncytial virus infected human dendritic cells by promoting Th1/Th17 responses.

PLoS One 2014 26;9(6):e100166. Epub 2014 Jun 26.

Dipartimento di Malattie Infettive, Parassitarie ed Immunomediate, Istituto Superiore di Sanità, Rome, Italy.

Respiratory Syncytial virus (RSV) is the leading cause of acute lower respiratory tract viral infection in young children and a major cause of winter hospitalization. Bordetella pertussis is a common cause of bacterial lung disease, affecting a similar age group. Although vaccines are available for B. pertussis infection, disease rates have recently increased in many countries. We have therefore developed a novel live attenuated B. pertussis strain (BPZE1), which has recently undergone a successful clinical phase I trial. In mice, BPZE1 provides protection against disease caused by respiratory viral challenge. Here, we analyze the effect of BPZE1 on antiviral T cell responses induced by human monocyte-derived dendritic cells (MDDC). We found that BPZE1 influences antiviral immune responses at several levels, enhancing MDDC maturation, IL-12p70 production, and shifting T cell cytokine profile towards a Th1/Th17 pattern. These data were supported by the intracellular signaling analysis. RSV infection of MDDC caused MyD88-independent STAT1 phosphorylation, whereas BPZE1 activated MyD88-dependent signaling pathways; co-infection caused both pathways to be activated. These findings suggest that BPZE1 given during infancy might improve the course and outcome of viral lung disease in addition to providing specific protection against B. pertussis infection.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0100166PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4072631PMC
February 2015

Chlamydia pneumoniae modulates human monocyte-derived dendritic cells functions driving the induction of a Type 1/Type 17 inflammatory response.

Microbes Infect 2013 Feb 16;15(2):105-14. Epub 2012 Nov 16.

Department of Infectious, Parasitic and Immune-Mediated Diseases, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy.

Chlamydia pneumoniae is a respiratory pathogen involved in the onset of chronic inflammatory pathologies. Dendritic cells (DC), are major players in spreading of C. pneumoniae from the lungs, a crucial step leading to disseminated infections. Less is known concerning modulation of DC functions consequent to encounter with the bacterium. In order to address this aspect, human monocyte-derived (MD)DC were infected with C. pneumoniae. After internalization bacterial counts increased in MDDC, as well as the expression of CPn1046, a gene involved in bacterial metabolism, with a peak 48 h after the infection. Infected MDDC switched to the mature stage, produced IL-12p70, IL-1β, IL-6, and IL-10, and drove a mixed Type 1/Type 17 polarization. Intracellular pathways triggered by C. pneumoniae involved Toll-like receptor (TLR) 2. Indeed, TLR2 was activated by C. pneumoniae in transfected HEK 293 cells, and C. pneumoniae-mediated phosphorylation of ERK1/2 was inhibited by an anti-TLR2 antibody in MDDC. When an ERK1/2 inhibitor was used, IL-12p70 and IL-10 release by MDDC was reduced and T cell polarization shifted towards a Type 2 profile. Overall, our findings unveiled the role played by TLR2 and ERK1/2 induced by C. pneumoniae to affect DC functions in a way that contributes to a Type 1/Type 17 pro-inflammatory response.
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http://dx.doi.org/10.1016/j.micinf.2012.11.004DOI Listing
February 2013

HIV-1 tat promotes integrin-mediated HIV transmission to dendritic cells by binding Env spikes and competes neutralization by anti-HIV antibodies.

PLoS One 2012 13;7(11):e48781. Epub 2012 Nov 13.

National AIDS Center, Istituto Superiore di Sanità, Rome, Italy.

Use of Env in HIV vaccine development has been disappointing. Here we show that, in the presence of a biologically active Tat subunit vaccine, a trimeric Env protein prevents in monkeys virus spread from the portal of entry to regional lymph nodes. This appears to be due to specific interactions between Tat and Env spikes that form a novel virus entry complex favoring R5 or X4 virus entry and productive infection of dendritic cells (DCs) via an integrin-mediated pathway. These Tat effects do not require Tat-transactivation activity and are blocked by anti-integrin antibodies (Abs). Productive DC infection promoted by Tat is associated with a highly efficient virus transmission to T cells. In the Tat/Env complex the cysteine-rich region of Tat engages the Env V3 loop, whereas the Tat RGD sequence remains free and directs the virus to integrins present on DCs. V2 loop deletion, which unshields the CCR5 binding region of Env, increases Tat/Env complex stability. Of note, binding of Tat to Env abolishes neutralization of Env entry or infection of DCs by anti-HIV sera lacking anti-Tat Abs, which are seldom present in natural infection. This is reversed, and neutralization further enhanced, by HIV sera containing anti-Tat Abs such as those from asymptomatic or Tat-vaccinated patients, or by sera from the Tat/Env vaccinated monkeys. Thus, both anti-Tat and anti-Env Abs are required for efficient HIV neutralization. These data suggest that the Tat/Env interaction increases HIV acquisition and spreading, as a mechanism evolved by the virus to escape anti-Env neutralizing Abs. This may explain the low effectiveness of Env-based vaccines, which are also unlikely to elicit Abs against new Env epitopes exposed by the Tat/Env interaction. As Tat also binds Envs from different clades, new vaccine strategies should exploit the Tat/Env interaction for both preventative and therapeutic interventions.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0048781PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3496724PMC
May 2013

Identity and ranking of colonic mesenchymal stromal cells.

J Cell Physiol 2012 Sep;227(9):3291-300

Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità, Rome, Italy.

Although ongoing clinical trials utilize systemic administration of bone-marrow mesenchymal stromal cells (BM-MSCs) in Crohn's disease (CD), nothing is known about the presence and the function of mesenchymal stromal cells (MSCs) in the normal human bowel. MSCs are bone marrow (BM) multipotent cells supporting hematopoiesis with the potential to differentiate into multiple skeletal phenotypes. A recently identified new marker, CD146, allowing to prospectively isolate MSCs from BM, renders also possible their identification in different tissues. In order to elucidate the presence and functional role of MSCs in human bowel we analyzed normal adult colon sections and isolated MSCs from them. In colon (C) sections, resident MSCs form a net enveloping crypts in lamina propria, coinciding with structural myofibroblasts or interstitial stromal cells. Nine sub-clonal CD146(+) MSC lines were derived and characterized from colon biopsies, in addition to MSC lines from five other human tissues. In spite of a phenotype qualitative identity between the BM- and C-MSC populations, they were discriminated and categorized. Similarities between C-MSC and BM-MSCs are represented by: Osteogenic differentiation, hematopoietic supporting activity, immune-modulation, and surface-antigen qualitative expression. The differences between these populations are: C-MSCs mean intensity expression is lower for CD13, CD29, and CD49c surface-antigens, proliferative rate faster, life-span shorter, chondrogenic differentiation rare, and adipogenic differentiation completely blocked. Briefly, BM-MSCs, deserve the rank of progenitors, whereas C-MSCs belong to the restricted precursor hierarchy. The presence and functional role of MSCs in human colon provide a rationale for BM-MSC replacement therapy in CD, where resident bowel MSCs might be exhausted or diverted from their physiological functions.
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http://dx.doi.org/10.1002/jcp.24027DOI Listing
September 2012

A combination HIV vaccine based on Tat and Env proteins was immunogenic and protected macaques from mucosal SHIV challenge in a pilot study.

Vaccine 2011 Apr 21;29(16):2918-32. Epub 2011 Feb 21.

National AIDS Center, Istituto Superiore di Sanità, Viale Regina Elena, 299 00161 Rome, Italy.

HIV native Tat and V2 loop-deleted Env (EnvΔV2) proteins already proved safe and immunogenic in phase I clinical testing as single vaccine components. Further, a phase II vaccine trial with Tat showed intensification of the therapeutic effects of HAART in successfully treated HIV-infected individuals. Here a pilot study assessed the immunogenicity and protective efficacy of an HIV/AIDS vaccine based on the combination of Tat and EnvΔV2 proteins in cynomolgus macaques against homologous intrarectal challenge with 35 MID(50) (monkey infectious dose 50) of an R5 simian-human immunodeficiency virus (SHIV(SF162P4cy)). Upon challenge, three of four macaques immunized with Tat and EnvΔV2, and two of three monkeys immunized with EnvΔV2 alone were protected from infection. In contrast, all three control animals, which had been either administered with the adjuvants only or left untreated, and an additional monkey immunized with Tat alone became systemically infected. Protection of the macaques vaccinated with EnvΔV2 or Tat/EnvΔV2 correlated with higher peak titers of pre-challenge neutralizing antibodies obtained during the immunization period (between 70 and 3 weeks before challenge) and with anti-Env V3 loop binding antibodies assessed 3 weeks before challenge. Compared to EnvΔV2 alone, the Tat and EnvΔV2 combined vaccine elicited faster antibody responses (IgM) with a trend, early in the vaccination schedule, after the second immunization including EnvΔV2, towards broader anti-Env IgG epitope specificity and a higher ratio of neutralizing to Env-binding antibody titers. As the number of immunizations increased, vaccination with EnvΔV2 approached the immune response assessed after two inocula with the Tat/EnvΔV2 combined vaccine, even though some differences remained between groups, as indicated by anti-Env IgG epitope mapping. In fact, three weeks before challenge, plasma IgG of animals in the EnvΔV2 group showed a trend towards stronger specificity for the V1 loop and V5 loop-C5 regions of Env, whereas the Tat/EnvΔV2 group displayed an overall higher reactivity for epitopes within the Env V3 loop throughout the immunization period. Although differences in terms of protection rate were not found between the EnvΔV2 or Tat/EnvΔV2 vaccination groups in this pilot study, vaccination with Tat/EnvΔV2 appeared to accelerate the induction of potentially protective antibody responses to Env. In particular, antibodies to the Env V3 loop, whose levels at pre-challenge correlated with protection, were already higher early in the vaccination schedule in monkeys immunized with Tat/EnvΔV2 as compared to EnvΔV2 alone. Further studies including larger vaccination groups and fewer immunizations with these two vaccine candidates are needed to confirm these findings and to assess whether the Tat/EnvΔV2 vaccine may afford superior protection against infection.
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http://dx.doi.org/10.1016/j.vaccine.2011.02.006DOI Listing
April 2011

Selective elimination of HIV-1-infected cells by Env-directed, HIV-1-based virus-like particles.

Virology 2006 Feb 4;345(1):115-26. Epub 2005 Nov 4.

AIDS Center, Istituto Superiore di Sanità, Viale Regina Elena, 299, 00161 Rome, Italy.

We recently showed that both replicating and resting cells cultivated with ganciclovir (GCV) were killed when challenged with vesicular stomatitis virus G glycoprotein pseudotyped HIV-1-based virus-like particles (VLPs) carrying the Nef7 (i.e., an HIV-1 Nef mutant incorporating in virions at high levels)/herpes simplex virus-1 thymidine kinase (HSV-TK) fusion product. On this basis, a novel anti-HIV therapeutic approach based on Nef7/TK VLPs expressing X4 or R5 HIV cell receptor complexes has been attempted. We here report that (CD4-CXCR4) and (CD4-CCR5) Nef7-based VLPs efficiently enter cells infected by X4- or R5-tropic HIV-1 strains, respectively. Importantly, the delivery of the VLP-associated Nef7/TK led to cell death upon GCV treatment. Of interest, VLPs were effective also against non-replicating, HIV-1-infected primary human monocyte-derived macrophages. HIV-targeted VLPs represent a promising candidate for the treatment of persistently HIV-1-infected cells that are part of virus reservoirs resistant to HAART therapies.
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http://dx.doi.org/10.1016/j.virol.2005.09.054DOI Listing
February 2006

Cell death induced by the herpes simplex virus-1 thymidine kinase delivered by human immunodeficiency virus-1-based virus-like particles.

Mol Ther 2005 Dec 10;12(6):1185-96. Epub 2005 Aug 10.

Department of Infectious, Parasitic, and Immune-Mediated Diseases, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy.

HIV-1 Nef incorporates into virions at low levels, likely about 10 molecules per viral particle. Here, we describe a Nef mutant (Nef7) apparently showing more than 100-fold higher efficiency of virion incorporation. Interestingly, Nef7 can act as a cargo molecule for protein delivery into the cells, as its virion incorporation appeared conserved even upon C-terminal fusion with proteins of up to 30 kDa. This was demonstrated first by assessing the intracellular fluorescence of cells challenged with lentivirus-based virus-like particles (VLPs) pseudotyped with the vesicular stomatitis virus envelope glycoprotein (VSV-G) and incorporating Nef7 fused with the green fluorescent protein. Furthermore, the biologic activity of products delivered by Nef7-based VLPs was demonstrated by tagging Nef7 with the herpes simplex virus-1 thymidine kinase (HSV-1 TK). In fact, we observed that both cell lines and primary human macrophages challenged with (VSV-G) Nef7/TK VLPs died after 5 to 7 days of treatment with ganciclovir (GCV). In sum, our findings support the notion that Nef7-based VLPs can be considered platforms for original systems of protein delivery. In particular, the here- described Nef7/TK VLPs represent a first applicative example opening the way toward new HSV-1 TK/GCV-based cell suicide therapies circumventing cell gene engineering procedures.
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http://dx.doi.org/10.1016/j.ymthe.2005.06.474DOI Listing
December 2005

HIV-1 Nef regulates the release of superoxide anions from human macrophages.

Biochem J 2005 Sep;390(Pt 2):591-602

Department of Infectious, Parasitic and Immune-mediated Diseases, Istituto Superiore di Sanità, viale Regina Elena 299, 00161 Rome, Italy.

The NADPH oxidase enzymatic complex participates in the oxidative burst by producing ROS (reactive oxygen species). Altered levels of ROS production may have pathogenetic implications due to the loss of some innate immune functions such as oxidative burst and phagocytosis. Considering that HIV-1 Nef protein plays a primary role in AIDS pathogenesis, by affecting the immune system, we sought to dissect possible effects of Nef on the release of superoxide anions. We show here that the inducible expression of Nef in human phagocytic cells modulates the superoxide release in a biphasic manner. In particular, an early Nef-induced increase of the superoxide release was followed by a dramatic decrease starting from 10 h after the Nef induction. This was observed whatever the presence of cell activators such as GM-CSF (granulocyte/macrophage colony-stimulating factor) or fMLP (N-formyl-L-methionyl-L-leucyl-L-phenylalanine). Whereas the early increase in superoxide release is probably the result of the already described Nef-dependent activation of PAK-2 (p21-activated kinase 2)-Rac2, we were interested in investigating the mechanisms underlying the late inhibition of superoxide release observed originally. In this regard, we individuated at least three independent requirements for the Nef-induced blockade of superoxide release: (i) the active protein synthesis; (ii) both the membrane localization and the interaction with endocytotic machinery of Nef; and (iii) the release of soluble factor(s). Moreover, we observed that IL-10 (interleukin-10) inhibits superoxide release, whereas its depletion restored NADPH oxidase activity. We propose that the cell membrane-to-lysosome Nef transit leads to the synthesis and release of soluble factor(s) and, among them, IL-10 might significantly contribute to the inhibition of NAPDH oxidase activity.
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http://dx.doi.org/10.1042/BJ20042139DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1198939PMC
September 2005

HIV-1 Nef enhances both membrane expression and virion incorporation of Env products. A model for the Nef-dependent increase of HIV-1 infectivity.

J Biol Chem 2004 May 19;279(22):22996-3006. Epub 2004 Mar 19.

Laboratory of Virology, Istituto Superiore di Sanità, Rome, 00161 Italy.

The expression of human immunodeficiency virus Nef increases the viral infectivity through mechanisms still not fully elucidated. Here we report that wild-type (wt) human immunodeficiency virus, type 1 (HIV-1), particles were neutralized by higher concentrations of either anti-Env glycoprotein (gp) 41 antibodies or recombinant soluble human CD4 compared with Deltanef HIV-1. This appeared to be the result of a Nef-induced increase of virion incorporation of both gp41 (transmembrane (TM)) and surface gp120 Env products likely originating from enhanced steady-state levels of cell membrane-associated Env products. This, in turn, seemed to be the consequence of a reduced retention of the Env precursor. Most interesting, we found that both the Nef-directed increase of Env membrane expression and the Nef-induced enhancement of HIV-1 infectivity relied on the presence of the intracytoplasmic domain of TM, supporting the hypothesis of a functional correlation between these effects. Mutagenesis studies allowed us to establish that the two leucine residues at the TM C terminus, which are part of a sorting motif involved in the control of Env membrane expression, and the 181-210-residue Nef C-terminal region were critically involved in the Nef/Env functional interaction. In conclusion, we propose that Nef increases the infectivity of HIV-1 at least in part by enhancing the amounts of Env products incorporated into virus particles.
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http://dx.doi.org/10.1074/jbc.M312453200DOI Listing
May 2004

HIV-1 Nef induces the release of inflammatory factors from human monocyte/macrophages: involvement of Nef endocytotic signals and NF-kappa B activation.

J Immunol 2003 Feb;170(4):1716-27

Laboratory of Virology, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161-Rome, Italy.

It has been recently reported that the endogenous expression of HIV-1 Nef in human monocyte/macrophages induces the release of chemokines and other as yet unidentified soluble factors leading to multiple effects of pathogenic significance, such as the recruitment and activation of quiescent lymphocytes. However, the description of underlying molecular mechanisms remained elusive. We recently demonstrated that human monocyte-derived macrophages (MDM) efficiently internalize soluble rNef, thereby inducing effects largely resembling those observed in cells endogenously expressing Nef. By exploiting the rNef/MDM model, we sought to gain more insights on the molecular mechanisms underlying the response of MDM to Nef. Array analysis for the detection of transcripts from a large number of monokines, chemokines, cytokines, and receptors thereof showed that MDM promptly responded to rNef treatment by increasing the transcription of genes for several inflammatory factors. Analysis of supernatants revealed that rNef treatment induced the release of macrophage inflammatory proteins 1alpha and 1beta, IL-1beta, IL-6, and TNF-alpha. Conversely, rNefs mutated in domains critical for the interaction with the endocytotic machinery (i.e., EE155-156QQ, and DD174-175AA) were ineffective. Interestingly, we found that the Nef-dependent release of inflammatory factors correlated with the activation of the NF-kappaB transcription factor, mainly in its p50/p50 homodimeric form, and in a de novo protein synthesis-independent manner. Our data add new hints supporting the idea that the presence of Nef is per se heavily detrimental for monocyte/macrophages and relative cross-talking cell types.
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http://dx.doi.org/10.4049/jimmunol.170.4.1716DOI Listing
February 2003

Inducible expression of the deltaNGFr/F12Nef fusion protein as a new tool for anti-human immunodeficiency virus type 1 gene therapy.

Hum Gene Ther 2002 Sep;13(14):1751-66

Laboratory of Virology, Istituto Superiore di Sanità, 00161 Rome, Italy.

Expression of the human immunodeficiency virus type 1 (HIV-1) Nef triple mutant F12Nef strongly inhibits HIV-1 replication. We exploited such a unique feature in a novel anti-HIV-1 gene therapy design by constructing an HIV-1 Tat-defective lentivirus vector expressing the product of fusion between the low-affinity human nerve growth factor receptor truncated in its intracytoplasmic domain (deltaNGFr, NH(2) moiety), and F12Nef (COOH moiety), under the control of the HIV-1 long terminal repeats. In this manner, both the selection marker (deltaNGFr) and the anti-HIV-1 effector are comprised in the same fusion protein, the expression of which is targetable by HIV-1 infection. Such a vector was proved to transduce human cells efficiently and, on HIV-1 infection, it expressed high levels of the fusion protein. In addition, strong antiviral activity of the deltaNGFr/F12Nef-expressing vector was demonstrated in cell lines as well as in primary cell cultures challenged with T- or M-tropic HIV-1 isolates. Thus, the HIV-1-targetable expression of the deltaNGFr/F12Nef fusion protein represents a novel and powerful tool for an effective anti-HIV-1 gene therapy strategy.
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http://dx.doi.org/10.1089/104303402760293583DOI Listing
September 2002