M. Vignoli

Effect of antibody to HIV-1 Tat protein on viral replication in vitro and progression of HIV-1 disease in vivo

In HIV-1-infected cell cultures, a relatively low concentration (5 micrograms/ml) of monoclonal antibody (mAb) against HIV-1-transactivating Tat protein was an efficient inhibitor of HIV-1 replication both in HIV-1(IIIB)-infected Jurkat cell and peripheral blood mononuclear cell (PBMC) cultures and significantly reduced the expression of a Tat-responsive CAT-reporter construct in HIV-1(IIIB)-infected Jurkat cells. Anti-Tat mAb also caused a significant reduction and a consistent delay in HIV-1 replication when added to PBMCs from HIV-1-infected patients cocultivated with phytohemagglutinin (PHA)-stimulated normal PBMCs. These data indicate that an autocrine-paracrine loop sustained by extracellular Tat protein, which is actively released by HIV-1-infected cells, may affect HIV-1 replication in cell cultures in vitro. An inverse relationship between natural anti-Tat antibody levels and p24 antigenemia was demonstrated by retrospective analysis of serial serum samples obtained from 10 HIV-1-seropositive hemophiliac patients followed over a 7-9-year period. This datum points to a possible influence of anti-Tat antibody on the progression of HIV-1 disease in vivo. These findings have strong implications for Tat protein as a possible target for specific immunotherapy in HIV-1-infected patients.

Antibodies against full-length Tat protein and some low-molecular-weight Tat-peptides correlate with low or undetectable viral load in HIV-1 seropositive patients

BACKGROUND The efficacy of a specific humoral response to transactivating Tat protein was studied in a group of HIV-1 seropositive drug addicts, who had previously received a similar course of anti-retroviral treatment with two reverse transcriptase inhibitors. OBJECTIVES The aim of the study was to evaluate the meaning of an immune response to Tat protein in HIV-1 seropositive patients with different levels of HIV-1 RNA viremia. STUDY DESIGN The study analyzed the presence of anti-Tat antibody reacting either with full-length Tat or with individual overlapping Tat-peptides (Tat(6-14), Tat(11-24), Tat(36-50), Tat(46-60), Tat(56-70) and Tat(65-80)), in a group of HIV-1 seropositive subjects with different peripheral blood viral loads. Plasma samples were examined by immunoenzymatic assay for the presence of anti-Tat IgG antibody and for the quantification of peripheral blood (plasma) viral load by branched DNA assay. RESULTS The large majority of HIV-1 patients showed detectable levels of serum IgG to full-length-Tat, and the anti-Tat antibody level presented an inverse correlation with viral load magnitude. The analysis of antibody levels against individual overlapping Tat-peptides clearly showed that an undetectable viral load was significantly associated with the presence of a high antibody concentration against Tat(6-14), Tat(36-50) and Tat(46-60) (P=0.002, P=0.027 and P<0.001, respectively). CONCLUSION In HIV-1-infected patients, a strong humoral immune response against HIV-1 Tat protein is inversely correlated to peripheral blood viral load and, in particular, a high level of antibody against Tat peptides containing amino acid residues 6-14 (Tat(6-14)), 36-50 (Tat(36-50)) and 46-60 (Tat(46-60)) is associated with an undetectable plasma viral load. These findings may help to tailor anti-HIV-1 Tat-containing vaccines.

An autocrine loop of HIV type-1 Tat protein responsible for the improved survival/proliferation capacity of permanently Tat-transfected cells and required for optimal HIV-1 LTR transactivating activity.

Human immunodeficiency virus type 1 (HIV-1) transactivating Tat protein is pivotal to virus replication. Tats potential effects on HIV-1 pathogenesis, however, go well beyond its role in the viruss life cycle. Current data indicate that biologically active Tat is released from HIV-1-infected cells and readily endocytosed and targeted to the nucleus of nearby, or perhaps distant, cells, where it may exert a series of pleiotropic effects. This paracrine action has been extensively investigated, and depending on the amounts of exogenously added Tat, its effects may extend from the suppression of immunocompetent cells to transactivation of heterologous genes to the promotion of growth of Kaposis sarcoma spindle cells. We have already observed that various cell lines, either permanently transfected with an expressive HIV-1 tat gene construct or cultured in the presence of exogenously added Tat protein, are protected from programmed cell death after serum withdrawal or other apoptotic stimuli. The present article shows that various types (lymphoblastoid, epithelial, neuronal) of permanently tat-transfected cell lines actively release fully bioactive Tat protein. The addition of anti-Tat antibody to the culture medium completely abolishes their increased survival/proliferation capacity in serum-free culture. In these conditions, therefore, the enhanced survival/proliferation potential of permanently tat-transfected cells seems entirely dependent on a Tat-protein autocrine loop. The finding that anti-Tat antibody, added to culture medium, exerts a negative influence on the expression of a Tat-responsive HIV-1 long terminal repeat chloramphenicol-acetyltransferase construct, transiently transfected into permanently tat-transfected cells, suggests that the Tat autocrine loop may also be required for optimal HIV-1 long terminal repeat transactivation.

Impaired telomerase activity in uninfected haematopoietic progenitors in HIV-1-infected patients

Objective: To evaluate the role of the selective forces exerted by the host on the HIV‐1 structures involved in viral entry. Design and methods: The V3 region of the env gene was analysed in cell‐free HIV‐1 RNA from 17 infected subjects: 11 long‐term non‐progressors (LTNP) and six symptomless, typical progressor patients. To evaluate the potential biological significance of one of the rare variants detected in the LTNP, it was reproduced by recombinant PCR into a HIV‐1 molecular clone. Results: The intrapatient divergence of the V3‐loop sequences averaged 8.62% in LTNP and 5.29% in progressors, although LTNP displayed lower divergence from the clade B consensus than progressors (16.65 and 19.76%, respectively). The analysis of non‐synonymous and synonymous substitutions indicated that selective pressure was exerted in this region in both LTNP and progressors. Individual peculiarities (unique and rare V3‐loop variants) emerged, however, in most sequences from LTNP, and variants bearing mutations in a domain crucial for the V3‐loop structure were more prevalent in LTNP (P = 0.0012). The pNL4–3‐derived mutant reproducing a V3‐loop variant detected in a LTNP was efficiently expressed upon transfection, but the mutant virus was nearly completely unable to infect CD4+ cell lines, activated primary peripheral blood lymphocytes, or monocyte‐derived macrophages, suggesting that a defect impaired the entry phase of the replication cycle. Conclusions: The results indicate that host factors impose selective constraints on the evolution of the HIV‐1 structures involved in viral entry. In LTNP, these factors are likely to force the virus into attenuated variants.Background: Haematopoietic progenitor cells (HPC) of HIV‐1‐infected patients are severely compromised in their replication and clonogenic capacities, and show an enhanced propensity to apoptosis, despite the lack of productive or latent HIV‐1 infection. Objective: To investigate telomerase enzyme levels in CD34+ HPC isolated from HIV‐1‐infected patients, because the absence of telomerase activity has been found to be correlated with a diminished replication potential. Methods: Telomerase levels were measured by a PCR‐based telomeric repeat amplification protocol. CD34+ HPC isolated from the peripheral blood of 11 HIV‐1‐infected patients were compared with CD34+ HPC isolated from peripheral blood (nine subjects) or bone marrow (six subjects) from 15 healthy donors. Telomerase levels were also studied in normal HPC after exposure to either gp120 or transforming growth factor (TGF)‐β1. Results: CD34+ HPC isolated from either peripheral blood or bone marrow from healthy donors expressed a high level of telomerase activity. On the contrary, CD34+ HPC isolated from HIV‐1‐seropositive patients did not express any detectable telomerase activity in nine patients, and a clearly reduced enzymatic activity in two patients. Furthermore, telomerase activity in normal CD34+ HPC exposed to recombinant gp120 was significantly reduced, and to a higher extent than in CD34+ HPC exposed to recombinant TGF‐β1. Conclusions: This is the first study to demonstrate severely impaired telomerase activity in uninfected CD34+ HPC isolated from HIV‐1‐infected patients. The mechanism underlying this impairment probably involves the interaction of HIV‐1 envelope glycoprotein gp120 with the cell membrane. These results may add to our understanding of the pathogenesis of the lesion of the HPC compartment.

In vitro infection of human epidermal Langerhans' cells with HIV-1

Human epidermal Langerhans cells (Lc) play an important role in antigen presentation and immunoregulation in the skin (for review see ref.(1)).They carry antigens and receptors which are identical to those of classical macrophages, i.e. FcIgG, C3 and the MHC class II HLA-D/DR antigens (2,3),but also the T-cell markers CDKT6) and CD4(T4) (4–6). Remarkably, Lc exhibit 50-fold higher amounts of HLA-DR than other cells of the monocyte-macrophage lineage (7).

High Levels of HIV-1 Replication Show a Clear Correlation With Downmodulation of Bcl-2 Protein in Peripheral Blood Lymphocytes of HIV-1-Seropositive Subjects

Peripheral blood lymphocytes (PBLs) from 51 HIV‐1‐seropositive subjects with different levels of HIV‐1 replication and 20 healthy blood donors were examined for the expression of the antiapoptotic Bcl‐2 protein. All the plasma samples from HIV‐1 patients were characterized for the presence of HIV‐1 p24 and HIV RNA viral load. Bcl‐2 protein expression in fresh peripheral blood lymphocytes was studied by different tests, including Western blot and indirect immunofluorescence techniques. Direct immunofluorescence staining, revealed by flow cytometry, was applied to quantify the number of specific anti‐Bcl‐2 antibody epitope binding sites, thus extrapolating the relative number of Bcl‐2 into the cells. The results indicate that the expression of Bcl‐2 protein is significantly lower in peripheral blood lymphocytes of HIV‐1‐seropositive patients showing high levels of viral replication, detected by means of HIV‐1 p24 and RNA viral load, with respect to HIV‐1 patients with low levels of virus replication and healthy blood donors. The clear‐cut inverse correlation between viral replication and Bcl‐2 expression reinforces the view that HIV‐1‐mediated apoptosis probably represents a key mechanism in AIDS pathogenesis. J. Med. Virol. 56:66–73, 1998.

Modulation of CD4, CXCR-4, and CCR-5 Makes Human Hematopoietic Progenitor Cell Lines Infected with Human Herpesvirus-6 Susceptible to Human Immunodeficiency Virus Type 1

Two CD34+ human hematopoietic progenitor cell (HPC) lines, KG-1 and TF-1, became susceptible to human immunodeficiency virus type 1 (HIV-1) infection in the presence of a concurrent infection by human herpesvirus-6 (HHV-6). We have analyzed the possible mechanism(s) underlying this phenomenon in light of the recent demonstration that at least two members of the chemokine receptor family, CXCR4 (LESTR/fusin) and CCR5 molecules, are the HIV-1-specific coreceptors necessary, together with the high-affinity receptor CD4, for entry into target cells of T-tropic and M-tropic HIV-1 isolates, respectively. KG-1 cells show CXCR4 and CCR5 surface molecules in a large proportion of the cell population. Therefore, their susceptibility to both T-tropic and M-tropic HIV-1 strains, caused by HHV-6 infection, can be explained by the HHV-6-induced appearance of CD4 molecules in about 40% of the cell population. In TF-1 cells, 10%-15% of which are CD4+ and exhibit a consistent CCR5 presence in a large proportion of the cell population and a hardly detectable amount of CXCR4 in a very limited number of cells, HHV-6 infection does not modify the cell surface availability of HIV-1-specific high-affinity receptor or coreceptors.

Enhanced nuclear factor-κB activation induced by tumour necrosis factor-α in stably tat-transfected cells is associated with the presence of cell-surface-bound Tat protein

Objective:An enhanced nuclear factor (NF)-κB activation in response to tumour necrosis factor (TNF)-α has been observed in stably tat-transfected cells. Recent experimental evidence suggests that Tat may autocrinously influence both cellular physiology and HIV-1 long terminal repeat-directed gene expression in Tat-producing cells. Therefore, the possible association of a Tat autocrinous loop with the enhanced NF-κB-binding activity induced by TNF-α in Tat-producing cells was studied by anti-Tat antibody blocking experiments. Design and methods:Permanently tat-transfected Jurkat cells, maintained either in the presence or absence of anti-Tat antibody, were studied for the presence of TNF-α-induced NF-κB-binding activity (quantified by electrophoretic mobility shift assays) and the presence of cell-surface-bound Tat (determined by flow cytometry and confocal microscopy of anti-Tat immunofluorescence-stained cell preparations). Results:The enhanced production of TNF-α-induced NF-κB binding activity exhibited by tat-transfected Jurkat cells was completely abolished in cell cultures maintained in the presence of anti-Tat antibody, thus indicating that the increased TNF-α-induced NF-κB binding activity observed in Jurkat-tat cells was dependent on the presence of Tat protein in an antibody-accessible location. In accordance with these findings, immunofluorescence-stained preparations of unfixed tat-transfected Jurkat cells showed the presence of cell-surface-bound Tat protein which was completely absent in cells incubated in the presence of anti-Tat antibodies. Conclusions:This study demonstrates that the enhanced NF-κB activation exhibited by stably tat-transfected cells in response to TNF-α, is associated with cell surface interaction of extracellularly released Tat protein. These data add further evidence to the possible relevance of a Tat autocrinous loop in the physiology of Tat-producing cells and suggest that in HIV-1-infected cells Tat is likely to behave as a bifunctional molecule which not only acts from within facilitating NF-κB recruitment in the viral transcription complex, but may also act from without increasing the availability of activated NF-κB.

Viral load trend in HIV-1 seropositive patients with different CD4 cell counts before starting HAART.

BACKGROUND The efficacy of highly active antiretroviral therapy (HAART) was prospectively monitored in HIV-1 seropositive patients by analyzing HIV-1 RNA viral load. OBJECTIVES The aim of the study was to evaluate the viral load course in two different groups of patients (group 1: CD4>400/mm(3), group 2: CD4<200/mm(3)) during a period ranging from 9 to 25 months. STUDY DESIGN HIV-1 viral load, at the start and during HAART, was analyzed in 117 patients who had previously been treated only with two anti-transcriptase drugs but were naive for protease inhibitors. RESULTS The results showed that, after the beginning of therapy, high plasma HIV-1 RNA levels dropped to undetectable values (<50 copies HIV-RNA/ml) in one third of patients over a mean period of about 9 months irrespective of the initial CD4 cell count, even though a viral reduction of at least 2log(s) in a significantly shorter period of time (P<0.001) was observed only among patients who began retroviral therapy with a higher CD4 cell count. CONCLUSION The response to HAART was not dramatically affected by the initial CD4 count. Though restricted to a small number of subjects, the data support the idea that therapeutic intervention can be effective even in an advanced stage of HIV-1 infection, when patients show a decreased number of CD4 T-lymphocytes.

Human T Cell Leukemia Virus Type II Increases Telomerase Activity in Uninfected CD34+ Hematopoietic Progenitor Cells

The aging process of long-term self-renewing hematopoietic stem/progenitor cells is not yet completely understood and recent studies on antiapoptotic cell pathways have demonstrated a close linkage between telomerase activation and Bcl-2 deregulation in human cancer cells. The present work shows that human T cell leukemia virus type II (HTLV-II) Mo virions that have originated from the T cell line (C344), but not from the B cell line (BJAB), are critically involved in mediating survival and growth effects on hematopoietic precursors (represented by both the TF-1 CD34+ cell line and by peripheral blood-derived CD34+ cells) through the maintenance or enhancement of telomerase activity and the induction of bcl-2 expression. In addition, using an interleukin-3-dependent TF-1 cell line, it was demonstrated that IL-3 deprivation was sufficient to influence the levels of telomerase activity and Bcl-2 expression in CD34+ cells. Taken together, these findings suggest that, in appropriate conditions, extended hematopoietic progenitor cell survival and proliferation following HTLV-II exposure depends on a synergistic interaction between up-regulation of Bcl-2 and activation of telomerase activity.