Barbara Ensoli

Control of SHIV-89.6P-infection of cynomolgus monkeys by HIV-1 Tat protein vaccine

Vaccine strategies aimed at blocking virus entry have so far failed to induce protection against heterologous viruses. Thus, the control of viral infection and the block of disease onset may represent a more achievable goal of human immunodeficiency virus (HIV) vaccine strategies. Here we show that vaccination of cynomolgus monkeys with a biologically active HIV-1 Tat protein is safe, elicits a broad (humoral and cellular) specific immune response and reduces infection with the highly pathogenic simian-human immunodeficiency virus (SHIV)-89.6P to undetectable levels, preventing the CD4+ T-cell decrease. These results may provide new opportunities for the development of a vaccine against AIDS.

HIV protease inhibitors are potent anti-angiogenic molecules and promote regression of Kaposi sarcoma.

Treatment with HIV-1 protease inhibitors (PI) is associated with a reduced incidence or regression of Kaposi sarcoma (KS). Here we show that systemic administration of the PIs indinavir or saquinavir to nude mice blocks the development and induces regression of angioproliferative KS-like lesions promoted by primary human KS cells, basic fibroblast growth factor (bFGF), or bFGF and vascular endothelial growth factor (VEGF) combined. These PIs also block bFGF or VEGF-induced angiogenesis in the chorioallantoic membrane assay with a potency similar to paclitaxel (Taxol). These effects are mediated by the inhibition of endothelial- and KS-cell invasion and of matrix metalloproteinase-2 proteolytic activation by PIs at concentrations present in plasma of treated individuals. As PIs also inhibit the in vivo growth and invasion of an angiogenic tumor-cell line, these data indicate that PIs are potent anti-angiogenic and anti-tumor molecules that might be used in treating non-HIV KS and in other HIV-associated tumors.

Biologically diverse molecular variants within a single HIV-1 isolate

AIDS is a disorder characterized by a slow progressive impairment of immune function and by infection of human immunodeficiency viruses (HIV-1, HIV-2)1–4. Our knowledge of how these viruses cause disease in man, or how the related lentiviruses (visna and equine infectious anaemia virus) cause disease in animals, is still fragmentary. In particular, the significance of genetic variation in HIV-1, occurring within populations, within individuals and over periods of time5,6, and the mechanisms of viral persistence remain unclear. To address these issues we prepared a series of proviral clones of HIV-1 originating from a single patient and compared their biological properties. Here we show that hybrid genomes (in which the envelope region of six viral clones were separately substituted into a prototype HIV-1 genome) generated viruses with widely differing capacity to grow in human T cells, cell lines and monocytoid cultures. These data suggest that extensive biological variation exists in vivo within an infected individual and is in part determined at the level of the viral envelope.

Kaposi’s sarcoma: a result of the interplay among inflammatory cytokines, angiogenic factors and viral agents

Kaposis sarcoma (KS) is an angioproliferative disease occurring in 4 clinic-epidemiologic forms. Although the AIDS-associated KS (AIDS-KS) is the most aggressive, all forms of KS share the same immunological and histopathological features suggesting common etiological and pathogenic factors. Recent data indicate that at least in early stage KS is not a real sarcoma but an angiohyperplastic-inflammatory lesion mediated by inflammatory cytokines and angiogenic factors, that is triggered or amplified by infection with human herpesvirus-8. In addition, the human immunodeficiency virus type-1 Tat protein appears to be responsible for the higher grade of aggressiveness of AIDS-KS as compared to the other forms of KS. However, given time, reactive KS may progress to a sarcoma as suggested by evidence of monoclonality in late-nodular lesions.

Native HIV-1 Tat Protein Targets Monocyte-Derived Dendritic Cells and Enhances Their Maturation, Function, and Antigen-Specific T Cell Responses

Vaccination of cynomolgus monkeys with the biologically active HIV-1 Tat protein induces specific Th1 responses, including CTLs. Similar responses are also induced by vaccination with tat DNA, but not by vaccination with inactivated Tat or Tat peptides. This suggested that the native Tat protein may act differently on APC as compared with inactivated Tat or peptide Ag. In this study, we show that biologically active Tat is very efficiently taken up by monocyte-derived dendritic cells (MDDC) in a time (within minutes)- and dose-dependent (starting from 0.1 ng/ml) fashion, whereas uptake is very poor or absent with other APC, including T cell blasts and B lymphoblastoid cell lines. Although maturation of MDDC reduces their pino/phagocytic activity, mature MDDC take up Tat much more efficiently than immature cells. In addition, Tat uptake is abolished or greatly hampered by oxidation/inactivation of the protein or by performing the experiments at 4°C, suggesting that MDDC take up native Tat by a receptor-mediated endocytosis. After uptake, active Tat protein induces up-regulation of MHC and costimulatory molecules and production of IL-12, TNF-α, and β chemokines, which drive Th1-type immune response. In contrast, these effects are lost by oxidation and inactivation of the protein. Finally, native Tat enhances Ag presentation by MDDC, increasing Ag-specific T cell responses. These data indicate that native Tat selectively targets MDDC, is taken up by these cells via specialized pathways, and promotes their maturation and Ag-presenting functions, driving Th1-type immune responses. Thus, Tat can act as both Ag and adjuvant, capable of driving T cell-mediated immune responses.

Vaccination with DNA containing tat coding sequences and unmethylated CpG motifs protects cynomolgus monkeys upon infection with simian/human immunodeficiency virus (SHIV89.6P)

Recent evidence suggests that a CD8-mediated cytotoxic T cell response against the Tat protein of human immunodeficiency virus (HIV)/simian immunodeficiency virus (SIV) controls primary infection after pathogenic virus challenge, and correlates with the status of long-term nonprogressor in humans. Due to the presence of unmethylated CpG sequences, DNA vaccination can boost the innate immunity driving more potent T cell-mediated immune responses. Therefore, cynomolgus monkeys were vaccinated with a tat-expressing vector containing defined unmethylated CpG sequences (pCV-tat). Here it is shown that the intramuscular inoculation of the pCV-tat contained primary infection with the highly pathogenic SHIV89.6P virus preventing the CD4(+) T cell decline in all the vaccinated monkeys. Undetectable virus replication and negative virus isolation correlated in all cases with the presence of anti-Tat CTLs. However, a CD8-mediated non cytolytic antiviral activity was also present in all protected animals. Of note, this activity was absent in the controls but was present in the monkey inoculated with the CpG-rich vector alone that was partially protected against viral challenge (i.e. no virus replication but positive virus isolation). These results suggest that a CTL response against Tat protects against primary infection by blocking virus replication at its early stage, in the absence of sterilizing immunity. Nevertheless, the boost of the innate immunity by CpG sequences can contribute to this protection both by driving more potent CTL responses and by inducing other CD8-mediated antiviral activities. Thus, the CpG-rich tat DNA vaccine may represent a promising candidate for preventive and therapeutic vaccination against AIDS.

The helical domain of GBP-1 mediates the inhibition of endothelial cell proliferation by inflammatory cytokines

Inflammatory cytokines (IC) activate endothelial cell adhesiveness for monocytes and inhibit endothelial cell growth. Here we report the identification of the human guanylate binding protein‐1 (GBP‐1) as the key and specific mediator of the anti‐proliferative effect of IC on endothelial cells. GBP‐1 expression was induced by IC, downregulated by angiogenic growth factors, and inversely related to cell proliferation both in vitro in microvascular and macrovascular endothelial cells and in vivo in vessel endothelial cells of Kaposis sarcoma. Experimental modulation of GBP‐1 expression demonstrated that GBP‐1 mediates selectively the anti‐proliferative effect of IC, without affecting endothelial cell adhesiveness for monocytes. GBP‐1 anti‐proliferative activity did not affect ERK‐1/2 activation, occurred in the absence of apoptosis, was found to be independent of the GTPase activity and isoprenylation of the molecule, but was specifically mediated by the C‐terminal helical domain of the protein. These results define GBP‐1 as an important tool for dissection of the complex activity of IC on endothelial cells, and detection and specific modulation of the IC‐activated non‐proliferating phenotype of endothelial cells in vascular diseases.

Use of HIV protease inhibitors to block Kaposi's sarcoma and tumour growth

HIV protease inhibitors are antiretroviral drugs that block the enzyme required for production of infectious viral particles. Although these agents have been designed to selectively bind to the catalytic site of HIV protease, evidence indicates that other cellular and microbial enzymes and pathways are also affected. It has been reported that patients treated with highly active anti-retroviral therapy (HAART) containing a protease inhibitor may be at reduced risk of Kaposis sarcoma (KS) and some types of non-Hodgkin lymphomas; some disease regressions have also been described. Here we review recent data showing that several widely used protease inhibitors, including indinavir, saquinavir, ritonavir, and nelfinavir, can affect important cellular and tissue processes such as angiogenesis, tumour growth and invasion, inflammation, antigen processing and presentation, cell survival, and tissue remodelling. Most of these non-HIV-related effects of protease inhibitors are due to inhibition of cell invasion and matrix metalloprotease activity, or modulation of the cell proteasome and NFkappaB. These elements are required for development of most tumours. Thus, by direct and indirect activities, protease inhibitors can simultaneously block several pathways involved in tumour growth, invasion, and metastasis. These findings indicate that protease inhibitors can be exploited for the therapy of KS and other tumours that occur in both HIV-infected and non-infected individuals. A multicentre phase II clinical trial with indinavir in non-HIV-associated KS is about to start in Italy.

Guanylate-Binding Protein-1 Expression Is Selectively Induced by Inflammatory Cytokines and Is an Activation Marker of Endothelial Cells during Inflammatory Diseases

During angiogenesis and inflammatory processes, endothelial cells acquire different activation phenotypes, whose identification may help in understanding the complex network of angiogenic and inflammatory interactions in vivo. To this goal we investigated the expression of the human guanylate-binding protein (GBP)-1 that is highly induced by inflammatory cytokines (ICs) and, therefore, may characterize IC-activated cells. Using a new rat monoclonal antibody raised against GBP-1, we show that GBP-1 is a cytoplasmic protein and that its expression in endothelial cells is selectively induced by interferon-gamma, interleukin-1alpha, interleukin-1beta, or tumor necrosis factor-alpha, but not by other cytokines, chemokines, or growth factors. Moreover, we found that GBP-1 expression is highly associated with vascular endothelial cells as confirmed by the simultaneous detection of GBP-1 and the endothelial cell-associated marker CD31 in a broad range of human tissues. Notably, GBP-1 expression was undetectable in the skin, but it was highly induced in vessels of skin diseases with a high-inflammatory component including psoriasis, adverse drug reactions, and Kaposis sarcoma. These results indicate that GBP-1 is a novel cellular activation marker that characterizes the IC-activated phenotype of endothelial cells.

Contribution of Nonneutralizing Vaccine-Elicited Antibody Activities to Improved Protective Efficacy in Rhesus Macaques Immunized with Tat/Env Compared with Multigenic Vaccines

Previously, chronic-phase protection against SHIV89.6P challenge was significantly greater in macaques primed with replicating adenovirus type 5 host range mutant (Ad5hr) recombinants encoding HIVtat and env and boosted with Tat and Env protein compared with macaques primed with multigenic adenovirus recombinants (HIVtat, HIVenv, SIVgag, SIVnef) and boosted with Tat, Env, and Nef proteins. The greater protection was correlated with Tat- and Env-binding Abs. Because the macaques lacked SHIV89.6P-neutralizing activity prechallenge, we investigated whether Ab-dependent cellular cytotoxicity (ADCC) and Ab-dependent cell-mediated viral inhibition (ADCVI) might exert a protective effect. We clearly show that Tat can serve as an ADCC target, although the Tat-specific activity elicited did not correlate with better protection. However, Env-specific ADCC activity was consistently higher in the Tat/Env group, with sustained cell killing postchallenge exhibited at higher levels (p < 0.00001) for a longer duration (p = 0.0002) compared with the multigenic group. ADCVI was similarly higher in the Tat/Env group and significantly correlated with reduced acute-phase viremia at wk 2 and 4 postchallenge (p = 0.046 and 0.011, respectively). Viral-specific IgG and IgA Abs in mucosal secretions were elicited but did not influence the outcome of the i.v. SHIV89.6P challenge. The higher ADCC and ADCVI activities seen in the Tat/Env group provide a plausible mechanism responsible for the greater chronic-phase protection. Because Tat is known to enhance cell-mediated immunity to coadministered Ags, further studies should explore its impact on Ab induction so that it may be optimally incorporated into HIV vaccine regimens.