The Sp1 transcription factor does not directly interact with the HIV-1 Tat protein.

The role of Sp1 in regulating the trans-activating activity of the human immunodeficiency virus type 1 (HIV-1) Tat protein has not yet been clearly defined. In fact, studies on the physical and functional interaction between Sp1 and Tat have yielded contradictory results. Here we investigated whether a physical interaction between Sp1 and Tat indeed occurs, exploiting both biochemical and genetic techniques that allow detection of direct protein-protein interactions. Studies performed with the yeast two-hybrid system indicate that Sp1 does not directly interact with the HIV-1 Tat protein. Control experiments demonstrated that both proteins are functionally expressed in the yeast cells. In vitro binding assays further confirmed that Sp1 does not physically bind Tat. These data suggest that in vivo Tat and Sp1 most likely take part of a multicomponent complex and thus encourage the search of the molecule(s) which mediate Tat-Sp1 interaction.

Immunization with low doses of HIV-1 tat DNA delivered by novel cationic block copolymers induces CTL responses against Tat.

Cytotoxic T cell responses are key to the control of intracellular pathogens including HIV-1. In particular, HIV-1 vaccines based on regulatory proteins, such as Tat, are aimed at controlling HIV-1 replication and at blocking disease development by inducing cytotoxic T cell responses. Naked DNA is capable of inducing such responses but it requires several inoculations of high amounts of DNA, and/or prime-boost regimens. Here, we show that a novel class of cationic block copolymers protect the DNA from DNAse I digestion, and improve DNA delivery to antigen-presenting cells (APCs) after intramuscular (i.m.) vaccination. In particular, three cationic block copolymers (K1, K2 and K5) were used to deliver the HIV-1 pCV-tat DNA vaccine in BALB/c mice. The results indicate that vaccination with a very low dose (1 microg) of pCV-tat delivered by the cationic block copolymer K2 is safe and, as compared to naked DNA (up to 30 microg), greatly increases the CTL response against Tat, which was detected in all animals in the absence or in the presence of re-stimulation.

Micellar-type complexes of tailor-made synthetic block copolymers containing the HIV-1 tat DNA for vaccine application.

A novel class of cationic block copolymers constituted by a neutral hydrophilic poly(ethylene glycol) (PEG) block and a positively charged poly(dimethylamino)ethyl methacrylate block was prepared for delivery of DNA. These block copolymers spontaneously assemble with DNA to give in aqueous medium micellar-like structures. Five of these novel block copolymers (K1-5), differing in the length of both the PEG chain and the linear charge density of the poly(dimethylamino)ethyl methacrylate block, were prepared and analyzed for gene delivery, gene expression and safety. All five block copolymers protected DNA from DNAse I digestion and delivered the DNA into the cell. However, only three of them (K1, K2 and K5) released the DNA at level allowing efficient gene expression into cells. No toxic effects of both the copolymers alone or their DNA complexes were observed in vitro or in mice. In addition, copolymers were scarcely immunogenic. These results indicate that this novel class of cationic block copolymers is safe and possesses the biological characteristics required for DNA delivery, thus, representing promising vehicles for DNA vaccination.