Long-term protection against HIV-1 infection conferred by tat or rev antisense RNA was affected by the design of the retroviral vector.

We have constructed a series of retroviral vectors in which the expression of antisense RNA targeted at the full length coding sequence of HIV-1 tat or rev was driven by three different promoters and in the context of double-copy or single-copy vectors. Jurkat cells transduced by these vectors were shown to express the expected tat or rev antisense RNA without alteration in cell proliferation or surface CD4 expression. After challenge with HIV, four patterns of protection were identified, with the degree of protection being determined primarily by the design of the expression system. In those patterns showing long-term complete protection, we could detect no HIV p24 in the culture supernatants or in the cells, and no HIV RNA or HIV proviral DNA (by PCR), during a 23-week follow-up. Experiments designed to rescue any live virus still formed in the culture after 20 weeks' challenge demonstrated that, with some constructs, infectious virus could no longer be isolated, while with other constructs, only a low level of infectious virus was still being formed and providing a continuing virus challenge, although all other markers of infection remained undetectable. Our results demonstrated that antisense RNA expression driven by tRNA promoter in the context of a double-copy vector conferred better long-term protection against HIV infection compared to that driven by HIV LTR or MLV LTR promoters, and that the optimized vectors may be useful in developing a gene therapy against HIV-1 infection and AIDS.