A human immunodeficiency virus type 1 pol gene-derived sequence (cPPT/CTS) increases the efficiency of transduction of human nondividing monocytes and T lymphocytes by lentiviral vectors.

We have investigated the capacity of two human immunodeficiency virus type 1-derived lentivectors, differing in the presence of a 118-bp pol fragment containing the cPPT/CTS element, to transduce human normal primary cells of different hematopoietic lineages. Infection of resting monocytes with a high multiplicity of infection (MOI > 10) revealed that the lentivirus carrying the pol fragment (cPPT) is effective, transducing 75% of cells compared with 36% for the no-cPPT vector. Even at low MOIs (< or =1) the cPPT vector still shows a better transduction efficiency than the no-cPPT vector. Moreover, transduction does not require dendritic cell differentiation. In contrast, infection of nonactivated T lymphocytes showed that both vectors, tested at high MOIs, can transduce a small, although measurable, percentage of cells (up to 10%), which may correspond to G(1a) "activated" cells as detected by simultaneous staining of DNA and RNA, in our cultures in the presence of medium alone. Furthermore, we show that the sole addition of interleukin 2 or interleukin 15 represents a full proliferative signal under our conditions and permits high transduction efficiency (up to 30% with the cPPT vector and 15% with the no-cPPT vector). Still higher transduction of T lymphocytes can be achieved after stimulation with phytohemagglutinin and interleukin 2 (up to 78% with the cPPT vector vs. 42% with the no-cPPT vector). Finally, both viruses do not transduce either resting or proliferating tonsillar B lymphocytes.

Selection of a T-cell line resistant to stavudine and zidovudine by prolonged treatment with stavudine.

It has been demonstrated that prolonged treatment with nucleoside analogues, such as 3'-azido-3'-deoxythymidine (zidovudine), 2',3'-dideoxycytidine (zalcitabine) and 9-(2-phosphonylmethoxyethyl) adenine (PMEA), may cause selection of cells that are resistant to their anti-HIV activity. A human T-lymphoblastoid cell line that is resistant to the antiviral and cytotoxic activity of 2',3'-didehydro-3'-deoxythymidine (stavudine) has developed as a result of prolonged treatment. These cells, called CEMstavudine, are also less sensitive to zidovudine. The cellular/pharmacological resistance acquired by the CEMstavudine cells is relatively low and appears to correlate with a reduction in thymidine kinase (TK) activity, rather than with a decreased expression of TK mRNA.

Lack of reduction of thymidine kinase activity in stavudine-treated HIV-infected patients.

It has been demonstrated that prolonged in vivo or in vitro treatment with some nucleosides analogs may favor the selection of cells with a reduced activity of enzymes involved in the phosphorylation of these drugs leading to a reduced sensitivity to their antiretroviral action. The aim of this study was to evaluate the effect, in vivo, of zidovudine and stavudine treatment on thymidine kinase (TK) activity. The results obtained showed that TK levels in PBMC from naive patients and stavudine-treated patients did not significantly differ (naive TK = 4.16 +/- 1.19 U/mg protein; stavudine TK = 3.65 +/- 1.73 U/mg protein; p = 0.42), suggesting that the treatment with this nucleoside analog is not associated to a defect of TK activity. On the contrary, PBMC from zidovudine-treated patients showed a significant reduction in TK activity compared to naive patients (naive TK = 4.16 +/- 1.19 U/mg protein; zidovudine TK = 2.70 +/- 1.54; p = 0.014. Although the clinical significance of these results has to be established, we can speculate that stavudine and zidovudine, which are presumably phosphorylated by the same cellular kinases, might display a different ability to in vivo select cells with a resistant phenotype.