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We have previously reported that a murine anti-Tat sFv intrabody, termed sFvtat1Ck, directed against the proline-rich N-terminal activation domain of HIV-1, is a potent inhibitor of HIV-1 replication [Mhashilkar, A. M., et al. (1995). EMBO J. 14, 1542-1551]. In this study, the protective effect of sFvtat1Ck expression on HIV-1 replication in both acutely infected and persistently infected CD4+ cells was examined. Stably transfected CD4+ SupT1 cells were resistant to HIV-1 infection at high MOI with both the laboratory isolate HxB2 and six syncytium-inducing (SI) primary isolates. Persistently infected U1 cells, which can be induced to increase HIV-1 mRNA synthesis on addition of PMA or TNF-alpha, showed decreased production of HIV-1 in the presence of sFvtat1Ck. In transduced CD4+-selected, CD8+-depleted, and total PMBCs, the sFvtat1Ck-expressing cells showed marked inhibition of HIV-1 replication. The anti-Tat sFv was subsequently humanized by substituting compatible human framework regions that were chosen from a large database of human V(H) and V(L) sequences on the basis of high overall framework matching, similar CDR length, and minimal mismatching of canonical and V(H)/V(L) contact residues. One humanized anti-Tat sFv intrabody, termed sFvhutat2, demonstrated a level of anti-HIV-1 activity that was comparable to the parental murine sFv when transduced PBMCs expressing the murine or humanized sFv intrabodies were challenged with HxB2 and two SI primary isolates. Because Tat is likely to have both direct and indirect effects in the pathogenesis of AIDS through its multiple roles in the HIV-1 life cycle and through its effects on the immune system, the strategy of genetically blocking Tat protein function with a humanized anti-Tat sFv intrabody may prove useful for the treatment of HIV-1 infection and AIDS, particularly when used as an adjuvant gene therapy together with highly active antiretroviral therapies that are currently available.

Human immunodeficiency virus type 1 (HIV-1) Tat, an early regulatory protein that is critical for viral gene expression and replication, transactivates the HIV-1 long terminal repeat (LTR) via its binding to the transactivation response element (TAR) and, along with other cellular factors, increases viral transcription initiation and elongation. Tat also superactivates the HIV-1 promoter through a TAR-independent mechanism, including tumor necrosis factor alpha-induced and protein kinase C (PKC)-dependent activation of NF-B, and inhibitors of Tat and NF-B cooperatively down-regulate this Tat-mediated LTR superactivation. In this study, a combined pharmacologic and genetic strategy using two PKC (NF-B) inhibitors, pentoxifylline (PTX) and Gö-6976, and a stably expressed anti-Tat single-chain intracellular antibody (sFv intrabody) was employed to obtain cooperative inhibition of both HIV-1 LTR-driven gene expression and HIV-1 replication. Treatment of cells with PTX and Gö-6976 resulted in cooperative inhibition of both HIV-1 LTR-driven gene expression and HIV-1 replication. In addition, the combined use of anti-Tat sFv intrabodies and the two NF-B inhibitorsretained the virus in the latent state for as long as 45 days. The combined treatment resulted in more durable inhibition of HIV-1 replication than was seen with the NF-B inhibitors alone or the anti-Tat sFv intrabodies alone. Together, these results suggest that in future clinical gene therapy trials, a combined pharmacologic and genetic strategy like the one reported here may improve the survival of transduced cells and prolong clinical benefit. Cells. The human CD4 ϩ T-lymphocyte cell lines SupT1 (SupT) and Jurkat were cultured in RPMI 1640 medium (Gibco/BRL, Gaithersburg, Md.) with glutamine (2 mM) supplemented with 10% fetal bovine serum at 37°C in an atmosphere of 5% CO 2 and 95% air. SupT cells stably transfected with pRc/ CMV vector (Invitrogen) (SupT-vector cells) or constitutively expressing the anti-HIV-1 Tat sFv intrabody termed sFvtat1Ck (SupT-sFvtat1Ck cells) were previously described (44) and were propagated in RPMI 1640 medium with G418 (Sigma Chemical Company, St. Louis, Mo.) at 500 g/ml.PBMCs were isolated from leukopacks (obtained from the Dana-Farber Cancer Institute Blood Bank) by Ficoll-Hypaque density gradient centrifugation. The CD4 ϩ mononuclear cells were selected from the total PBMC population with anti-CD4-coated cell culture flasks (GenCell, Santa Clara, Calif.). A total of 20 ϫ 10 6 PBMCs were resuspended in 4 ml of Ca 2ϩ -and Mg 2ϩ -free phosphatebuffered saline (PBS/CMF)-1 mM EDTA-0.5% human gamma globulins (Sigma) and incubated on the flask for 1 h at room temperature. The flask was washed five times with PBS/CMF-1 mM EDTA. After the final wash, 10 ml of RPMI 1640 medium supplemented with 10% fetal calf serum and phytohemagglutinin (PHA) (1 g/ml) was added to the flask, which was then incubated for circa 60 h at 37°C with 5% CO 2 .Reagents and plasmids. PTX [1-(5Ј-oxohexyl)-3,7-dimethyl xanthine] and Gö-6976 (a synthetic nonglyc...