Francesca Vitone

Quantitative detection of human immunodeficiency virus type 1 (HIV-1) proviral DNA in peripheral blood mononuclear cells by SYBR green real-time PCR technique

BACKGROUND The persistence of proviral human immunodeficiency virus type 1 (HIV-1) DNA reservoir represents one of the major drawbacks to the total eradication of HIV-1. The quantitative determination of proviral HIV-1 DNA load offers significant therapeutic information, especially when the HIV-1 RNA levels drop below the detectable limits during the highly active retroviral therapy (HAART) treatment. Moreover, the detection of HIV-1 proviral DNA is an important diagnostic marker in the evaluation of HIV-1 infection of newborns of HIV-1 seropositive women. OBJECTIVE We evaluated a real-time PCR based on LightCycler technology revealed through SYBR green fluorochrome (SYBR green real-time PCR) to quantify the HIV-1 proviral DNA load in peripheral blood mononuclear cells (PBMC) of HIV-1 seropositive patients. STUDY DESIGN Firstly, we assessed the SYBR green real-time quantitative PCR for HIV-1 proviral DNA load detection determining the specificity and sensitivity of the assay using the LightCycler system. Secondly, we tested the performance of our SYBR green real-time PCR on 50 HIV-1 seropositive patients under HAART and 20 blood donors. RESULTS/CONCLUSIONS The results of this study showed that our SYBR green real-time PCR is able to detect five copies of the HIV-1 genome. Moreover, our method revealed HIV-1 proviral DNA in all the 50 HIV-1 seropositive patients ( 627 +/- 1068 HIV-1 proviral DNA copies per 10(6) PBMC, with a range of 30-6300 copies), whereas no positive signal was observed in any PBMC blood donors. Our SYBR green real-time PCR represents a sensitive and useful approach that could be applied both in HIV-1 proviral DNA reservoir determination and in HAART monitoring, particularly when the HIV-1 plasmatic RNA is undetectable.

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.

HIV-1 Tat protein concomitantly down-regulates apical caspase-10 and up-regulates c-FLIP in lymphoid T cells: a potential molecular mechanism to escape TRAIL cytotoxicity.

In this study, we showed the existence of a positive correlation between the amount of human immunodeficiency virus‐type 1 (HIV‐1) RNA in HIV‐1 seropositive subjects and the plasma levels of TRAIL. Since it has been previously demonstrated that HIV‐1 Tat protein up‐regulates the expression of TRAIL in monocytic cells whereas tat‐expressing lymphoid cells are more resistant to TRAIL cytotoxicity, we next investigated the effect of Tat on the expression/activity of both apical caspase‐8 and ‐10, which play a key role in mediating the initial phases of apoptosis by TRAIL, and c‐FLIP. Jurkat lymphoblastoid human T cell lines stably transfected with a plasmid expressing wild‐type (HIV‐1) tat gene showed normal levels of caspase‐8 but significantly decreased levels of caspase‐10 at both mRNA and protein levels with respect to Jurkat transfected with the control plasmid or with a mutated (cys22) non‐functional tat cDNA. A significant decrease of caspase‐10 expression/activity was also observed in transient transfection experiments with plasmid carrying tat cDNA. Moreover, c‐FLIPL and c‐FLIPS isoforms were up‐regulated in tat‐expressing cells at both mRNA and protein level in comparison with control cells. Taken together, these results provide a molecular basis to explain the resistance of tat‐expressing Jurkat cells to apoptosis induced by TRAIL and, possibly, to other death‐inducing ligands.

HIV-1 DNA load analysis in peripheral blood lymphocytes and monocytes from naïve and HAART-treated individuals

OBJECTIVE To evaluate HIV-1 DNA load in PBLs and monocytes from both long-term HAART-treated and antiretroviral naïve HIV-1 infected patients. METHODS Cross-sectional quantitative analysis of HIV-1 DNA load was performed in PBLs and monocytes, purified from 34 long-term HAART-treated and 34 naïve HIV-1 infected patients, and compared to RNA viral load and CD4+ cell count. RESULTS HAART-treated patients showed significantly lower levels of viral DNA both in PBLs and monocytes in comparison with naïve individuals. Variable levels of HIV-1 DNA amount in monocytes were detected in all naïve patients but only in 12 of 34 HAART-treated individuals. PBLs HIV-1 DNA load was inversely correlated to CD4+ cell count in naïve and HAART-treated patients whereas no association was detected in monocytes. CONCLUSIONS Long-term HAART decreased HIV-1 DNA load in PBLs and monocytes demonstrating a valuable inhibitor effect, especially in short-lived reservoirs. In addition, the positive correlation of DNA burden between PBLs and monocytes may suggest a dynamic relation between these reservoirs in the course of disease. HIV-1 DNA load quantitative analysis in PBLs and monocytes may be considered an important approach to study the HIV-1 reservoir and the effectiveness of HAART therapy in HIV-1 seropositive patients.

HIV-1 negatively affects the survival/maturation of cord blood CD34+ hematopoietic progenitor cells differentiated towards megakaryocytic lineage by HIV-1 gp120/CD4 membrane interaction

To investigate the mechanisms involved in the human immunodeficiency virus type 1 (HIV‐1)‐related thrombocytopenia (TP), human umbilical cord blood (UCB) CD34+ hematopoietic progenitor cells (HPCs) were challenged with HIV‐1IIIb and then differentiated by thrombopoietin (TPO) towards megakaryocytic lineage. This study showed that HIV‐1, heat‐inactivated HIV‐1, and HIV‐1 recombinant gp120 (rgp120) activated apoptotic process of megakaryocyte (MK) progenitors/precursors and decreased higher ploidy MK cell fraction. All these inhibitory effects on MK survival/maturation and platelets formation were elicited by the interaction between gp120 and CD4 receptor on the cell membrane in the absence of HIV‐1 productive infection. In fact, in our experimental conditions, HPCs were resistant to HIV‐1 infection and no detectable productive infection was observed. We also evaluated whether the expression of specific cytokines, such as TGF‐β1 and APRIL, involved in the regulation of HPCs and MKs proliferation, was modulated by HIV‐1. The specific protein and mRNA detection analysis, during TPO‐induced differentiation, demonstrated that HIV‐1 upregulates TGF‐β1 and downregulates APRIL expression through the CD4 engagement by gp120. Altogether, these data suggest that survival/differentiation of HPCs committed to MK lineage is negatively affected by HIV‐1 gp120/CD4 interaction. This long‐term inhibitory effect is also correlated to specific cytokines regulation and it may represent an additional mechanism to explain the TP occurring in HIV‐1 patients. J. Cell. Physiol. 210: 315–324, 2007.

Selective up-regulation of functional CXCR4 expression in erythroid cells by HIV-1 Tat protein

CXCR4 is the high affinity receptor for the SDF‐1α chemokine and represents the main coreceptor for HIV‐1 T‐tropic strains. The surface expression of CXCR4 was analysed in CD34+ haematopoietic progenitors, induced to differentiate along the erythroid or granulocytic lineages, in liquid cultures supplemented or not with HIV‐1 Tat protein. At concentrations as low as 1–10 ng/ml, synthetic Tat protein significantly increased the surface expression of CXCR4 in erythroid but not in granulocytic cells. The Tat‐mediated up‐regulation of surface CXCR4 was accompanied by a concomitant increase of CXCR4 mRNA and total CXCR4 protein content in cells developing along the erythroid lineage after 6–10 days of culture. Moreover, addition of SDF‐1α (200 ng/ml) induced a significant higher rate of apoptosis in Tat‐treated erythroid cells in comparison with control cells. These results demonstrated for the first time a direct positive role in haematopoietic gene regulation of Tat protein, and suggest the possible involvement of Tat in HIV‐1‐induced anaemia.

Molecular and phylogenetic analysis of HIV-1 variants circulating in Italy

ObjectiveThe continuous identification of HIV-1 non-B subtypes and recombinant forms in Italy indicates the need of constant molecular epidemiology survey of genetic forms circulating and transmitted in the resident population.MethodsThe distribution of HIV-1 subtypes has been evaluated in 25 seropositive individuals residing in Italy, most of whom were infected through a sexual route during the 1995–2005 period. Each sample has been characterized by detailed molecular and phylogenetic analyses.Results18 of the 25 samples were positive at HIV-1 PCR amplification. Three samples showed a nucleotide divergence compatible with a non-B subtype classification. The phylogenetic analysis, performed on both HIV-1 env and gag regions, confirms the molecular sub-typing prediction, given that 1 sample falls into the C subtype and 2 into the G subtype. The B subtype isolates show high levels of intra-subtype nucleotide divergence, compatible with a long-lasting epidemic and a progressive HIV-1 molecular diversification.ConclusionThe Italian HIV-1 epidemic is still mostly attributable to the B subtype, regardless the transmission route, which shows an increasing nucleotide heterogeneity. Heterosexual transmission and the interracial blending, however, are slowly introducing novel HIV-1 subtypes. Therefore, a molecular monitoring is needed to follow the constant evolution of the HIV-1 epidemic.

HIV‐1 DNA proviral load in treated and untreated HIV‐1 seropositive patients

As proviral human immunodeficiency virus type 1 (HIV-1) DNA can replenish and revive viral infection upon activation, its detection might offer significant therapeutic information, complementing the input provided by plasma RNA determination in the follow-up of infected individuals. A selected group of acutely infected subjects was studied to verify both total and 2-long terminal repeat (2-LTR) DNA proviral load during the acute phase of infection and thereafter. Patients were divided in two sex- and age-matched groups: 19 naive individuals who did not receive antiretroviral therapy during the observation period and 20 subjects treated according to current guidelines. Total and 2-LTR HIV-1 DNA proviral load, in addition to RNA viral load and CD4 cell count, were determined in peripheral blood mononuclear cells (PBMC) at baseline, 6 and 12 months after the first sampling. Total and 2-LTR HIV-1 DNA proviral load exhibited no significant variation at any time in the naive patients (total HIV-1 DNA ranging from 896 + or - 731 to 715 + or - 673 copies/10(5) PBMC and 2-LTR HIV-1 DNA ranging from 94 + or - 105 to 65 + or - 44 copies/10(5) PBMC), whereas a significant reduction in both total HIV-1 DNA (ranging from 997 + or - 676 to 262 + or - 174 copies/10(5) PBMC) and 2-LTR HIV-1 DNA proviral load (ranging from 116 + or - 55 to 26 + or - 35 copies/10(5) PBMC) was detected in highly active antiretroviral therapy (HAART) patients, together with a CD4(+) T cell count increase and RNA load decrease. HAART negatively affects both the labile HIV burden and the integrated proviral DNA, at least in the initial period of successful treatment, suggesting that quantification of HIV-1 DNA proviral load may be an important parameter in monitoring HIV infection.

Mutation patterns of the reverse transcriptase genes in HIV-1 infected patients receiving combinations of nucleoside and non nucleoside inhibitors

A genotyping assay was used to define human immunodeficiency virus type 1 (HIV-1) reverse transcriptase codons in plasma samples from 80 HIV-1 patients extensively treated with two nucleoside reverse transcriptase (zidovudine and lamivudine) and one non nucleoside reverse transcriptase (nevirapine) inhibitor. The frequencies of T215S/Y/F, M41L, D67N, L210W K70R, K219Q mutations, detectable in plasma samples, conferring resistance to zidovudine were 61.2, 56.2, 36.2, 31.5, 27.5 and 17.5%, respectively. Mutations (M184V or M184I) conferring resistance to lamivudine were detected in an extremely high percentage of patients (61%). Among mutations correlated to high (K103N, V106A, Y181C/I, Y188C/H/L, G190A/C/E/Q/S/T) or moderate (V108I, V118I) levels of nevirapine resistance, the predominant amino acid change was a substitution at 103 codon, present in 24 of 80 samples tested. Finally Q151M, the marker mutation able to confer resistance to all nucleoside analogues, was detected in seven patients with a viral load of between 1 x 10(4) and 9 x 10(4) HIV-1 RNA copies/ml. The relationship between the genotype and the viral load showed that the incidence of some specific mutations [M41L, T215Y (correlated to zidovudine resistance) and K103N (correlated to all NNRTIs drugs)] significantly (P=0.001) increased with higher viral load. Our results, albeit limited to a small cohort, showed a high frequency of mutations correlated to drugs in use, suggesting a need for therapeutic change in the near future and demonstrating that the development of genotyping tests helps to guide the therapeutic management of HIV-1 infected people. Our data highlight the dangers of selecting antiretroviral therapy without previous antiretroviral drug testing. Although the cost of these assays is a concern, prescribing inefficacious drugs could create serious problems for HIV-1 patients.

Development of drug resistance in HIV-1 patients receiving a combination of stavudine, lamivudine and efavirenz

The study evaluated the development of drug resistance in a group of HIV-1 patients. After failure to respond to previous therapy with two non-nucleoside reverse transcriptase inhibitors (NNRTIs), as assessed by the presence of a rebound in viral load or a constant high level of HIV plasma viraemia, the patients were treated with a combination of stavudine, lamivudine and efavirenz (EFV). Results showed that viruses carrying primary mutations, usually K103N, T215Y and M41L, presented higher levels of HIV-1 RNA, suggesting an association between a precise mutation pattern and treatment failure.