In vitro evolution of a highly replicating, doxycycline-dependent HIV for applications in vaccine studies

A major concern associated with the use of vaccines based on live-attenuate d viruses is the possible and well documented reversion to pathogenic pheno types. In the case of HIV, genomic deletions or mutations introduced to att enuate viral pathogenicity can be repaired by selection of compensating mut ations. These events lead to increased virus replication rates and, eventua lly, disease,progression. Because replication competence and degree of,prot ection appear to be directly correlated, further attenuation of a vaccine v irus may compromise the ability to elicit a protective immune response. Her e, we describe an approach toward a safe attenuated HIV vaccine. The system is not based on permanent reduction of infectivity by alteration of import ant viral genomic sequences, but on strict control of replication through t he insertion of the tetracycline (Tet) system in the HIV genome. Furthermor e, extensive in vitro evolution was applied to the prototype Tet-controlled HIV to select for variants with optimized rather than diminished replicati on capacity. The final product of evolution has properties uniquely suited for use as a vaccine strain. The evolved virus is highly infectious, as opp osed to a canonically attenuated virus. It replicates efficiently in T cell lines and in activated and unstimulated peripheral blood mononuclear cells . Most importantly, replication is strictly dependent on the nontoxic Tet-a nalogue doxycycline and can be turned on and off. These results suggest tha t this in vitro evolved, doxycycline-dependent HIV might represent a useful tool toward the development of a safer, live-attenuated HIV vaccine.