Inhibition of human immunodeficiency virus type 1 replication in vitro in acutely and persistently infected human CD4(+) mononuclear cells expressingmurine and humanized anti-human immunodeficiency virus type 1 Tat single-chain variable fragment intrabodies

We have previously reported that a murine anti-Tat sFv intrabody, termed sF vtat1Ck, directed against the proline-rich N-terminal activation domain of HIV-1, is a potent inhibitor of HIV-1 replication [Mhashilkar, A, M., et nl , (1995), EMBO J, 14, 1542-1551], In this study, the protective effect of s Fvtat1Ck expression on HIV-1 replication in both acutely infected and persi stently infected CD4(+) cells was examined, Stably transfected CD4(+) SupT1 cells were resistant to HIV-1 infection at high MOI with both the laborato ry isolate HxB2 and six syncytium-inducing (SI) primary isolates. Persisten tly infected U1 cells, which can be induced to increase HIV-1 mRNA synthesi s 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 substitu ting compatible human framework regions that were chosen from a large datab ase of human VH and VL sequences on the basis of high overall framework mat ching, 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 pare ntal murine sFv when transduced PBMCs expressing the murine or humanized sF v intrabodies were challenged with HxB2 and two SI primary isolates. Becaus e Tat is likely to have both direct and indirect effects in the pathogenesi s of AIDS through its multiple roles in the HIV-1 life cycle and through it s effects on the immune system, the strategy of genetically blocking Tat pr otein 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 ad juvant gene therapy together with highly active antiretroviral therapies th at are currently available.