Potent and stable attenuation of live-HIV-1 by gain of a proteolysis-resistant inhibitor of NF-kappa B (I kappa B-alpha S32/36A) and the implicationsfor vaccine development

Live-attenuated human immunodeficiency viruses (HIVs) are candidates for Ac quired Immunodeficiency Syndrome (AIDS) vaccine. Based on the simian immuno deficiency virus (SIV) model for AIDS, loss-of-function (e.g. deletion of a ccessory genes such as nef) has been forwarded as a primary approach for cr eating enfeebled, but replication-competent, HIV-1/SIV. Regrettably, recent evidence suggests that loss-of-function alone is not always sufficient to prevent the emergence off virulent mutants. New strategies that attenuate v ia mechanisms distinct from loss-of-function are needed for enhancing the s afety phenotype of viral genome. Here, we propose gain-of-function to be us ed simultaneously with loss-of-function as a novel approach for attenuating HIV-1, We have constructed an HIV-1 genome carrying the cDNA of a proteoly sis-resistant nuclear factor-kappa B inhibitor (I kappa B-alpha S32/36A) in the nef region. HIV-1 expressing 1 kappa B-alpha S32/36A down-regulates vi ral expression and is highly attenuated in both Jurkat and peripheral blood mononuclear cells. We provide formal proof that the phenotypic and attenua ting characteristics of I kappa B-alpha S32/36A permit its stable maintenan ce in a live, replicating HIV-1 despite 180 days of forced ex vivo passagin g in tissue culture. As compared with other open-reading frames embedded in to HIV/SIV genome, this degree of stability is unprecedented. Thus, I kappa B-alpha S32/36A offers proof-of-principle that artifactually gained functi ons, when used to attenuate the replication of live HIV-1, can be stable. T hese findings illustrate gain-of-function as a feasible strategy for develo ping safer live-attenuated HIVs to be tested as candidates for AIDS vaccine .