Extracellular Vesicles and Their Use as Vehicles of Immunogens.

Healthy cells constitutively release lipid bilayered vesicles of different sizes and recognizing different biogenesis, collectively referred to as extracellular vesicles (EVs). EVs can be distinguished in exosomes and microvesicles. Biological and biomedical research on EVs is an emerging field that is rapidly growing. Many EV features including biogenesis, cell uptake, and functions still require unambiguous elucidation. Nevertheless, it has been well established that EVs are involved in communication among cells, tissues, and organs under both healthy and disease conditions by virtue of their ability to deliver macromolecules to target cells. Here, we summarize most recent findings regarding biogenesis, structure, and functions of both exosomes and microvesicles. In addition, the use of EVs as delivery tools to induce CD8+ T cell immunity is addressed compared to current designs exploiting enveloped viral vectors and virus-like particles. Finally, we describe a both safe and original approach conceived for the induction of strong CTL immunity against antigens uploaded in EVs constitutively released by muscle cells.

High HIV-1 diversity in immigrants resident in Italy (2008-2017).

The proportion of new diagnoses of HIV infection in immigrants residing in Italy raised from 11% in 1992 to 29.7% in 2018. To investigate the HIV clades circulating in this community a retrospective study was performed in 557 HIV-infected immigrants living in 12 Italian cities. Immigrants originated from East-Europe and Central-Asia (11.7%), North Africa and Middle East (7.3%), South and South-East Asia (7.2%), Latin America and the Caribbean (14.4%), and sub-Saharan Africa (59.4%). More than 87% of immigrants were on antiretroviral therapy (ART), although 26.6% of them were viremic. A 22.0% of immigrants had hepatitis (HBV and/or HCV) and/or tuberculosis. HIV phylogenetic analysis on sequences from 192 immigrants showed the presence of clades B (23.4%), G (16.1%), C (10.4%), A1 (9.4%), F1 (5.2%), D (1.6%) and Circulating Recombinant Forms (CRFs) (33.9%). CRF02_AG represented 72.3% of the total CRFs. Clusters between immigrants and Italian natives were also present. Drug resistance mutations to NRTI, NNRTI, and PI drug classes occurred in 29.1% of ART-treated and in 12.9% of ART-naïve individuals. These data highlight the need for tailored public health interventions in immigrants to avoid spreading in Italy of HIV genetic forms and ART-resistant variants, as well as HIV co-morbidities.

Neurodegenerative Disorders Treatment: The MicroRNA Role.

Neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, Huntington's disease and prion disease are not timely and effectively treated using conventional therapies. This emphasizes the need for alternative therapeutic approaches. In this respect, gene-based therapies have been adopted as potentially feasible alternative therapies, where the microRNA (miRNA) approach has experienced a great explosion in recent years. Because miRNAs have been shown to be implicated in the pathogenesis of several diseases including neurodegenerative diseases, they are intensely studied as candidates for diagnostic and prognostic biomarkers, as predictors of drug response and as therapeutic agents. In this review, we evaluate the feasibility of both direct and indirect miRNA mimics and inhibitors toward the regulation of neurodegenerative-related genes both in vivo and in vitro models, highlight the advantages and drawbacks associated with miRNA-based therapy, and summarize the relevant techniques and approaches attempted to deliver miRNAs to the central nervous system for therapeutic purposes, with particular regard to the exosomes. Additionally, we describe a new approach that holds great promise for the treatment of a wide range of diseases including neurodegenerative disorders. This approach is based on addressing the incorporation of miRNAs into exosomes to increase the quantity and quality of miRNA packed and delivered to the central nervous system and other sites of action.

The CD8⁺ T Cell-Mediated Immunity Induced by HPV-E6 Uploaded in Engineered Exosomes Is Improved by ISCOMATRIXTM Adjuvant.

We recently described the induction of an efficient CD8⁺ T cell-mediated immune response against a tumor-associated antigen (TAA) uploaded in engineered exosomes used as an immunogen delivery tool. This immune response cleared tumor cells inoculated after immunization, and controlled the growth of tumors implanted before immunization. We looked for new protocols aimed at increasing the CD8⁺ T cell specific response to the antigen uploaded in engineered exosomes, assuming that an optimized CD8⁺ T cell immune response would correlate with a more effective depletion of tumor cells in the therapeutic setting. By considering HPV-E6 as a model of TAA, we found that the in vitro co-administration of engineered exosomes and ISCOMATRIXTM adjuvant, i.e., an adjuvant composed of purified ISCOPREPTM saponin, cholesterol, and phospholipids, led to a stronger antigen cross-presentation in both B- lymphoblastoid cell lines ( and monocyte-derived immature dendritic cells compared with that induced by the exosomes alone. Consistently, the co-inoculation in mice of ISCOMATRIXTM adjuvant and engineered exosomes induced a significant increase of TAA-specific CD8⁺ T cells compared to mice immunized with the exosomes alone. This result holds promise for effective usage of exosomes as well as alternative nanovesicles in anti-tumor therapeutic approaches.

HPV-E7 delivered by engineered exosomes elicits a protective CD8⁺ T cell-mediated immune response.

We developed an innovative strategy to induce a cytotoxic T cell (CTL) immune response against protein antigens of choice. It relies on the production of exosomes, i.e., nanovesicles spontaneously released by all cell types. We engineered the upload of huge amounts of protein antigens upon fusion with an anchoring protein (i.e., HIV-1 Nefmut), which is an inactive protein incorporating in exosomes at high levels also when fused with foreign proteins. We compared the immunogenicity of engineered exosomes uploading human papillomavirus (HPV)-E7 with that of lentiviral virus-like particles (VLPs) incorporating equivalent amounts of the same antigen. These exosomes, whose limiting membrane was decorated with VSV-G, i.e., an envelope protein inducing pH-dependent endosomal fusion, proved to be as immunogenic as the cognate VLPs. It is noteworthy that the immunogenicity of the engineered exosomes remained unaltered in the absence of VSV-G. Most important, we provide evidence that the inoculation in mouse of exosomes uploading HPV-E7 induces production of anti-HPV E7 CTLs, blocks the growth of syngeneic tumor cells inoculated after immunization, and controls the development of tumor cells inoculated before the exosome challenge. These results represent the proof-of-concept about both feasibility and efficacy of the Nefmut-based exosome platform for the induction of CD8+ T cell immunity.

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

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.

The reverse transcription inhibitor abacavir shows anticancer activity in prostate cancer cell lines.

BACKGROUND: Transposable Elements (TEs) comprise nearly 45% of the entire genome and are part of sophisticated regulatory network systems that control developmental processes in normal and pathological conditions. The retroviral/retrotransposon gene machinery consists mainly of Long Interspersed Nuclear Elements (LINEs-1) and Human Endogenous Retroviruses (HERVs) that code for their own endogenous reverse transcriptase (RT). Interestingly, RT is typically expressed at high levels in cancer cells. Recent studies report that RT inhibition by non-nucleoside reverse transcriptase inhibitors (NNRTIs) induces growth arrest and cell differentiation in vitro and antagonizes growth of human tumors in animal model. In the present study we analyze the anticancer activity of Abacavir (ABC), a nucleoside reverse transcription inhibitor (NRTI), on PC3 and LNCaP prostate cancer cell lines. PRINCIPAL FINDINGS: ABC significantly reduces cell growth, migration and invasion processes, considerably slows S phase progression, induces senescence and cell death in prostate cancer cells. Consistent with these observations, microarray analysis on PC3 cells shows that ABC induces specific and dose-dependent changes in gene expression, involving multiple cellular pathways. Notably, by quantitative Real-Time PCR we found that LINE-1 ORF1 and ORF2 mRNA levels were significantly up-regulated by ABC treatment. CONCLUSIONS: Our results demonstrate the potential of ABC as anticancer agent able to induce antiproliferative activity and trigger senescence in prostate cancer cells. Noteworthy, we show that ABC elicits up-regulation of LINE-1 expression, suggesting the involvement of these elements in the observed cellular modifications.

Generation of a human immunodeficiency virus type 1 chronically infected monkey B cell line expressing low levels of endogenous TRIM5alpha.

Several innate cellular antiviral factors exist in mammalian cells that prevent the replication of retroviruses. Among them, the tripartite motif protein (TRIM)5alpha has been shown to block human immunodeficiency virus type 1 (HIV-1) infection in several types of Old World monkey cells. Here we report a novel HIV-1 chronically infected monkey B cell line, F6/HIV-1, characterized by very low levels of TRIM5alpha expression that allows HIV-1 to overcome the restriction. Virus produced by F6/HIV-1 cells fails to infect monkey cells but retains the ability to infect human peripheral blood mononuclear cells (PBMCs) and T cell lines, although with a reduced infectivity compared to the input virus. Ultrastructural analyses revealed the presence of budding virions at the F6/HIV-1 cells plasma membrane characterized by a typical conical core shell. To our knowledge F6/HIV-1 is the first monkey cell line chronically infected by HIV-1 and able to release infectious particles thus representing a useful tool to gain further insights into the molecular mechanisms of HIV-1 pathogenesis.

IRF-1 is required for full NF-kappaB transcriptional activity at the human immunodeficiency virus type 1 long terminal repeat enhancer.

Human immunodeficiency virus type 1 (HIV-1) gene expression is controlled by a complex interplay between viral and host factors. We have previously shown that interferon-regulatory factor 1 (IRF-1) is stimulated early after HIV-1 infection and regulates promoter transcriptional activity even in the absence of the viral transactivator Tat. In this work we demonstrate that IRF-1 is also required for full NF-kappaB transcriptional activity. We provide evidence that IRF-1 and NF-kappaB form a functional complex at the long terminal repeat (LTR) kappaB sites, which is abolished by specific mutations in the two adjacent kappaB sites in the enhancer region. Silencing IRF-1 with small interfering RNA resulted in impaired NF-kappaB-mediated transcriptional activity and in repressed HIV-1 transcription early in de novo-infected T cells. These data indicate that in early phases of HIV-1 infection or during virus reactivation from latency, when the viral transactivator is absent or present at very low levels, IRF-1 is an additional component of the p50/p65 heterodimer binding the LTR enhancer, absolutely required for efficient HIV-1 replication.

Tat protein vaccination of cynomolgus macaques influences SHIV-89.6P cy243 epitope variability.

In a previous study we showed that vaccination with the native Tat protein controlled virus replication in five out of seven monkeys against challenge with the simian human immunodeficiency virus (SHIV)-89.6P cy243 and that this protection correlated with T helper (Th)-1 response and cytotoxic T lymphocyte (CTL) activity. To address the evolution of the SHIV-89.6P cy243 both in control and vaccinated infected monkeys, the sequence of the human immunodeficiency virus (HIV)-1 Tat protein and the C2-V3 Env region of the proviral-DNA-derived clones were analyzed in both control and vaccinated but unprotected animals. We also performed analysis of the T cell epitope using a predictive epitope model taking into consideration the phylogeny of the variants. Our results suggest that even though the viral evolution observed in both groups of monkeys was directed toward variations in the major histocompatibility complex (MHC)-I epitopes, in the control animals it was associated with mutational escape of such epitopes. On the contrary, it is possible that viral evolution in the vaccinated monkeys was linked to mutations that arose to keep high the viral fitness. In the vaccinated animals the reduction of epitope variability, obtained prompting the immune system by vaccination and inducing a specific immunological response against virus, was able to reduce the emergence of escape mutants. Thus the intervention of host's selective forces in driving CTL escape mutants and in modulating viral fitness appeared to be different in the two groups of monkeys. We concluded that in the vaccinated unprotected animals, vaccination with the Tat protein induced a broad antiviral response, as demonstrated by the reduced ability to develop escape mutants, which is known to help in the control of viral replication.

Infection of a simian B cell line by human and simian immunodeficiency viruses.

It is well known that HIV-1 does not establish infection in nonhuman primates, nor in cell lines derived from them, due to the existence of saturable resistance factors. In this study, we show that an in vitro established Macaca fascicularis-derived CD4(-) B cell line (F6) can be productively infected by the laboratory-adapted T-tropic HXBc2/HIV-1 strain at low multiplicity of infection, apparently because it does not express the restriction factor that has been detected in other simian cell lines. Moreover, efficient entry into F6 cells was obtained with pseudotyped recombinant HIV-1 viruses containing the laboratory-adapted T-tropic (HXBc2) or the dual-tropic (89.6) envelope glycoproteins, whereas entry of virus containing the envelope glycoproteins of the M-tropic Ba-L strain was less efficient. Virus containing primary T-tropic (Eli) envelope glycoproteins did not infect F6 cells. Furthermore, although CCR5 was not present on the cell surface and gpr15 and strl33 mRNAs were not expressed in the cells, a high level of infection of F6 cells by the M-tropic simian immunodeficiency virus SIVmac316 was observed. In contrast, F6 cells were poorly infected by T-tropic SIVmac239. Given the unique properties of the F6 cell line, i.e., that it is of simian origin yet is able to be infected by HIV-1 in a CD4-independent manner, F6 cells represent a useful model for studying cellular factors mediating resistance or permissivity to HIV-1 infection and may help to evaluate HIV-1 and SIV cell tropism.

Long-term protection against SHIV89.6P replication in HIV-1 Tat vaccinated cynomolgus monkeys.

Vaccination with a biologically active Tat protein or tat DNA contained infection with the highly pathogenic SHIV89.6P virus, preventing CD4 T-cell decline and disease onset. Here we show that protection was prolonged, since neither CD4 T-cell decline nor active virus replication was observed in all vaccinated animals that controlled virus replication up to week 104 after the challenge. In contrast, virus persisted and replicated in peripheral blood mononuclear cells and lymph nodes of infected animals, two of which died. Tat-specific antibody, CD4 and CD8 T-cell responses were high and stable only in the animals controlling the infection. In contrast, Gag-specific antibody production and CD4 and CD8 T-cell responses were consistently and persistently positive only in the monkeys that did not control primary virus replication. These results indicate that vaccination with Tat protein or DNA induced long-term memory Tat-specific immune responses and controlled primary infection at its early stages allowing a long-term containment of virus replication and spread in blood and tissues.

Analysis of the signal transduction pathway leading to human immunodeficiency virus-1-induced interferon regulatory factor-1 upregulation.

Interferon (IFN) regulatory factors (IRFs) constitute a family of transcriptional activators and repressors involved in the regulation of immune system, host defense, and cell growth. All members share conserved DNA-binding domains that recognize DNA sequences termed IRF-binding elements/IFN-stimulated response elements (IRF-E/ISRE) present on the promoter of IFN-alpha/beta and IFN-stimulated genes. An ISRE has been identified downstream of the transcription start site of the long terminal repeat (LTR) of human immunodeficiency virus-1 (HIV-1). Our previous results showed that among the IRF factors, IRF-1 is able to stimulate HIV-1 LTR transcription and its expression is induced by HIV-1, early, upon infection and before the expression of Tat. In this study we investigated the signal transduction pathway leading to HIV-1-induced IRF-1 expression. Key IRF-1 promoter elements that mediate the activation of transcription upon induction by inflammatory cytokines are IFN-gamma-activated sequences that bind members of the signal transducer and activator of transcription (STAT) family and binding sites for nuclear factor kappaB (NF-kappaB). Both STAT-1 and NF-kappaB activation were examined to determine putative molecular targets whose inhibition resulted in the inhibition of HIV-1 replication. The results show that at early time points after HIV-1 infection, NF-kappaB but not STAT-1 is activated. Moreover, a significant decrease in HIV-1 replication was observed upon de novo infection of Jurkat T cells expressing an NF-kappaB super-repressor (IkappaB-alpha 2NDelta4). These results suggest that in early phases of HIV-1 infection, before detectable cytokine production, NF-kappaB seems responsible for HIV-1-induced IRF-1 expression.

On the role of interferon regulatory factors in HIV-1 replication.

Interferons (IFNs) are pleiotropic cytokines that possess several biological activities and play a central role in basic and applied research as mediators of antiviral and antigrowth responses, modulators of the immune system, and therapeutic agents against viral diseases and cancer. Interferon regulatory factors (IRFs) have been identified together with signal transducers and activators of transcription (STAT) from studies on the type I IFN as well as IFN-stimulated (ISG) gene regulation and signaling. IRFs constitute a family of transcriptional activators and repressors implicated in multiple biological processes including regulation of immune responses and host defence, cytokine signaling, cell growth regulation, and hematopoietic development. All members share a well-conserved DNA binding domain at the NH(2)-terminal region that recognizes similar DNA sequences, termed IRF element (IRF-E)/interferon-stimulated response element (ISRE), present on the promoter of target genes. Recently, a sequence homologous to the ISRE has been identified downstream from the 5' human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR). This sequence is a binding site for IRF-1 and IRF-2. Here we briefly summarize the role of IRFs in the regulation of HIV-1 LTR transcriptional activity and virus replication. The overall effect of IRFs on HIV-1 replication will also be discussed in the context of strategies carried out by the virus to counteract the IFN-mediated host defences both in active replication and during the establishment of viral latency.

HIV-1 Tat-based vaccines: from basic science to clinical trials.

Vaccination against human immunodeficiency virus (HIV)-1 infection requires candidate antigen(s) (Ag) capable of inducing an effective, broad, and long-lasting immune response against HIV-1 despite mutation events leading to differences in virus clades. The HIV-1 Tat protein is more conserved than envelope proteins, is essential in the virus life cycle and is expressed very early upon virus entry. In addition, both humoral and cellular responses to Tat have been reported to correlate with a delayed progression to disease in both humans and monkeys. This suggested that Tat is an optimal target for vaccine development aimed at controlling virus replication and blocking disease onset. Here are reviewed the results of our studies including the effects of the Tat protein on monocyte-derived dendritic cells (MDDCs) that are key antigen-presenting cells (APCs), and the results from vaccination trials with both the Tat protein or tat DNA in monkeys. We provide evidence that the HIV-1 Tat protein is very efficiently taken up by MDDCs and promotes T helper (Th)-1 type immune responses against itself as well as other Ag. In addition, a Tat-based vaccine elicits an immune response capable of controlling primary infection of monkeys with the pathogenic SHIV89.6P at its early stages allowing the containment of virus spread. Based on these results and on data of Tat conservation and immune cross-recognition in field isolates from different clades, phase I clinical trials are being initiated in Italy for both preventive and therapeutic vaccination.

Modulation of human immunodeficiency virus 1 replication by interferon regulatory factors.

Transcription of the human immunodeficiency virus (HIV)-1 is controlled by the cooperation of virally encoded and host regulatory proteins. The Tat protein is essential for viral replication, however, expression of Tat after virus entry requires HIV-1 promoter activation. A sequence in the 5' HIV-1 LTR, containing a binding site for transcription factors of the interferon regulatory factors (IRF) family has been suggested to be critical for HIV-1 transcription and replication. Here we show that IRF-1 activates HIV-1 LTR transcription in a dose-dependent fashion and in the absence of Tat. This has biological significance since IRF-1 is produced early upon virus entry, both in cell lines and in primary CD4+ T cells, and before expression of Tat. IRF-1 also cooperates with Tat in amplifying virus gene transcription and replication. This cooperation depends upon a physical interaction that is blocked by overexpression of IRF-8, the natural repressor of IRF-1, and, in turn is released by overexpression of IRF-1. These data suggest a key role of IRF-1 in the early phase of viral replication and/or during viral reactivation from latency, when viral transactivators are absent or present at very low levels, and suggest that the interplay between IRF-1 and IRF-8 may play a key role in virus latency.

Rapid methods for identification of poliovirus isolates and determination of polio neutralizing antibody titers in human sera.

A new rapid method for identification and determination of the titer of polioviruses and other enteroviruses in cell monolayers grown in microtiter plates is described. The method is based on immunoperoxidase staining of infected cells with commercial monoclonal antibodies (MAb) and biotin-labeled secondary antibody. The presence of poliovirus or other enteroviruses was established as the appearance of at least one focus of cells with stained cytoplasm 6 h post-infection. Viral titers determined by this method were expressed as focus forming unit (FFU) per ml which was found to correspond approximately to 10 TCID(50)/ml. The suitability of this technique to determine poliovirus antibody titers in human sera was also tested comparing the immunocytochemical neutralization assay with a conventional neutralization in microtiter plates. The test was standardized using reference human sera in order to produce antibody titers expressed in international units (IU). In addition to high reproducibility, the new neutralization test appears to be sensitive, specific and rapid, and might thus represent a useful tool for the diagnosis of polio and other enterovirus infections.

Mapa de soluções da Estratégia Saúde da Família Caminhando com a Saúde

A pesquisa realizada na área de abrangência da ESF "Caminhando Com a Saúde" partiu do pressuposto que, apesar das várias divulgações a respeito da hipertensão artéria sistêmica (HAS) e seus fatores de risco e a importância as consulta trimestral para controle e renovação de receita, as pessoas portadoras dessa enfermidade, ainda apresentam sérias dificuldades em lidar com a mesma e que tais dificuldades são decorrentes de questões culturais e sociais. O presente estudo apresenta, em sua primeira parte, um histórico sobre a HAS, seus fatores de risco, o tratamento e a responsabilidade da equipe de saúde; em sua segunda parte expõe a metodologia da pesquisa e na terceira parte, situa a HAS dentro da área de abrangência da ESF "Caminhando Com a Saúde". Através desta pesquisa, observamos ainda que o controle da pressão arterial não se relaciona apenas aos hábitos de vida saudável do paciente e seu tratamento medicamentoso, mas também com a conscientização sobre a enfermidade e às comorbidades relacionadas. Deve-se enfatizar o trabalho de conscientização e de orientação do uso correto de medicações conforme prescrição, desenvolvida pela ESF "Caminhando Com a Saúde" e da importância da consulta trimestral