Giuliano Furlini

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.

Recombinant human immunodeficiency virus type-1 (HIV-1) Tat protein sequentially up-regulates IL-6 and TFG-β1 mRNA expression and protein synthesis in peripheral blood monocytes

Summary. In this study we evaluated the effect of human immunodeficiency virus type 1 (HIV‐1) recombinant Tat protein on mRNA expression and protein synthesis of two inflammatory cytokines ‐ interleukin‐6 (IL‐6) and transforming growth factor‐β1 (TGF‐β1) ‐ by peripheral blood (PB) monocytes. Whereas maximal levels of IL‐6 protein were recovered in PB monocyte culture supernatants after 24–48 h from the addition of 1 μg/ml of recombinant Tat, TGF‐β1 showed a slower and progressive increase, reaching maximal levels only after 72–96h of culture. Consistently, the analysis of the steady‐state levels of mRNA showed a sharp increse of IL‐6 mRNA expression after 24h of culture, with a slow decline thereafter. On the other hand, TGF‐β1 mRNA expression showed a slow increase only after 72–96h of culture. Moreover, IL‐6 appeared involved in the up‐regulation of TGF‐β1, because the addition of a neutralizing anti‐IL‐6 antibody to Tat‐treated PB monocyte cultures significantly reduced the amounts of TGF‐β1 recovered in the culture supernatants after 96 h.

Hepatitis C virus core antigen: Analytical performances, correlation with viremia and potential applications of a quantitative, automated immunoassay

BACKGROUND Testing for hepatitis C virus core antigen (HCV Ag) may represent a complementary tool to anti-HCV and HCV-RNA in the diagnosis and monitoring of HCV infection. OBJECTIVE To evaluate the performance characteristics of the automated Abbott ARCHITECT HCV Ag assay. STUDY DESIGN Five sites analyzed over 3000 routine serum samples from populations at different risk, comparing HCV Ag results with anti-HCV screening and supplemental assay results and with HCV-RNA. RESULTS The HCV Ag assay showed a specificity of 100%, a good precision (CV<10%) and excellent dilution linearity (r>0.999). The sensitivity (3 fmol/L) corresponds to 700-1100 IU/mL of HCV-RNA. A non-linear correlation with HCV-RNA was found: r=0.713 vs. Siemens bDNA (523 specimens), r=0.736 vs. Roche Cobas TaqMan (356 specimens) and r=0.870 vs. Abbott Real-Time PCR (273 specimens). HCV Ag quantitation was equally effective on different HCV genoypes (239 for genotype 1/1a/1b/1c, 108 for genotype 2/2a/2c, 86 for genotype 3/3a, 50 for genotype 4/4a/4c/4d). Testing of subjects at high risk for HCV and with potential or actual impairment of the immune system identified 2 cases negative for anti-HCV and positive for HCV Ag on 361 hemodialyzed (0.6%) and 7 cases on 97 (7.2%) among transplant recipients. HCV Ag positivity anticipated anti-HCV seroconversion in all three cases of acute hepatitis C. CONCLUSIONS HCV Ag may be used as reflex testing on anti-HCV positive individuals to confirm or exclude an active infection, and on subjects with acute hepatitis or belonging to high risk groups.

Uninfected haematopoietic progenitor (CD34+) cells purified from the bone marrow of AIDS patients are committed to apoptotic cell death in culture

ObjectiveTo determine the mechanism underlying the poor growth in vitro of haematopoietic progenitor cells isolated from HIV-1-infected patients. MethodApoptotic death in liquid culture of bone-marrow CD34+ cells obtained from 11 HIV-1-seropositive patients and 18 HIV-1-seronegative donors was quantitatively monitored by a flow cytometry procedure. ResultsNo significant differences in the percentage of apoptotic cells were noted between the two groups immediately after purification. When CD34 + cells were placed in liquid cultures supplemented with 2 ng/ml interleukin-3, the number of apoptotic cells progressively and significantly (P < 0.05) increased in all HIV-1-seropositive patients, while it remained constant in HIV-1-seronegative individuals. Although all HIV-1-seropositive patients showed signs of active viral replication in the bone-marrow micro-environment, progenitor CD34 + cells did not show the presence of active and/or latent HIV-1 infection. ConclusionOur data demonstrate that CD34 + cells isolated from AIDS patients with active HIV-1 replication in bone-marrow accessory cells are committed to apoptotic death without being directly affected by productive infection.

Human immunodeficiency virus type 1 envelope glycoprotein gp120-mediated killing of human haematopoietic progenitors (CD34+ cells).

The effects of human immunodeficiency virus type 1 (HIV-1) and recombinant envelope glycoprotein gp120 on the in vitro growth of enriched human haematopoietic progenitors (CD34+ cells) have been investigated. A 2 h exposure to HIV-1 resulted in a progressive and significant reduction of viable CD34+ cell number in liquid cultures and of granulocyte-macrophage, erythroid and megakaryocytic progenitors in semisolid cultures. In virus-treated CD34+ cells, no signs of active virus replication were observed and the possibility of latent infection was excluded by quantitative polymerase chain reaction. Recombinant HIV-1 envelope glycoprotein gp120 added to CD34+ cell cultures displayed a dose-dependent inhibitory activity on CD34+ cell viability. Neutralizing antibody against gp120 was able to block completely the inhibitory activity on CD34+ cells of either HIV-1 or recombinant gp120. These results demonstrate that HIV-1 envelope glycoprotein gp120 has a direct cytotoxic effect on CD34+ cells.

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.

Inhibitory effect of HIV-1 envelope glycoproteins gp120 and gp160 on the in vitro growth of enriched (CD34+) hematopoietic progenitor cells.

SummaryThe effect of increasing concentrations (from 0.01 to 10 µg/ml) of HIV-1 envelope glycoproteins gp160, gp120, gp41 and core protein p24 was evaluated on the in vitro growth of enriched hematopoietic progenitors (CD34+ cells). Both gp120 and gp160, at concentrations from 0.01 to 10 µg/ml, caused a progressive and significant (p<0.05) decrease in viable CD34+ cell count in liquid cultures supplemented with 2 ng/ml of human recombinant (r) interleukin-3 (IL-3), evaluated by means of Trypan-blue exclusion and [3H]thymidine ([3H]TdR) incorporation. In the absence of rIL-3, no inhibitory effects were observed even at the highest gp160 and gp120 concentrations explored (10 µg/ml). On the contrary, gp41 and p24 did not affect the number of viable CD34+ cells, either in the presence or in the absence of rIL-3. Moreover, gp160 and gp120, but not gp41 and p24, significantly (p<0.05) inhibited the in vitro growth of granulomacrophage progenitors (CFU-GM) in a dose-dependent fashion. These data clearly demonstrate that HIV-1 envelope glycoproteins inhibit the growth of purified hematopoietic progenitors. We propose that HIV-1 can impair hematopoiesis through the interaction of gp120/gp160 with CD34+ cell surface, independently of an infectious process.

Human immunodeficiency virus type 1 (HIV-1) and human hematopoietic progenitor cells.

SummaryBesides a progressive depletion of CD4+ T-lymphocytes, other peripheral blood cytopenias, (granulocytopenia, anemia and thrombocytopenia) are frequently observed in HIV-1 seropositive individuals, especially in patients with overt AIDS. Various experimental evidences suggest that HIV-1 could play a direct role in the pathogenesis of HIV-1 related peripheral blood cytopenias, affecting the survival/proliferation capacity of hematopoietic progenitors. CD34+ human hematopoietic progenitors, however, are substantially not susceptible to HIV-1 infection either in vitro and in vivo and their defects seem rather related to an alteration of bone marrow and peripheral blood microenvironments due to the presence of soluble HIV-1 specific products.

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.