Enhancement of antigen specific humoral immune responses after delivery of a DNA plasmid based vaccine through a contact-independent helium plasma.

Non-viral in vivo delivery of DNA, encoding for specific proteins, has traditionally relied on chemical or physical forces applied directly to tissues. Physical methods typically involve contact between an applicator/electrode and tissue and often results in transient subject discomfort. To overcome these limitations of contact-dependent delivery, a helium plasma source was utilized to deposit ionized gasses to treatment/vaccination sites without direct contact between the applicator and the tissues. The study reported here evaluated the efficacy of this strategy as an effective method to administer DNA vaccines. Balb/C mice were vaccinated with a DNA plasmid expressing an HIVgp120 envelope glycoprotein either with or without co-administration of helium plasma or electroporation. The results indicated, for the first time, the potential efficacy of helium plasma delivery for the induction and enhancement of antigen specific immune responses following DNA vaccination.

Anti-cancer activity of the HIV accessory molecule viral protein R (Vpr): Delivery as a DNA expression plasmid or biologically active peptides.

By virtue of its ability to induce cell cycle arrest and apoptosis, the HIV accessory protein Vpr (viral protein R) has been evaluated by us and others as an anti-proliferative/anti-cancer agent. We have demonstrated that Vpr, when delivered to established experimental B16.F10 melanoma tumors in mice as a DNA expression plasmid through in vivo electroporation, can result in complete regression of the established tumors. We have also demonstrated that Vpr peptides from the carboxy region of the protein can inhibit in vitro growth of both B16.F10 melanoma as well as human HeLa cervical carcinoma tumor cells. These findings, summarized in this report, underscore the potential of Vpr as an anti-cancer agent and warrants, we believe, further experimental as well as clinical evaluation.

Analysis of the potential for HIV-1 Vpr as an anti-cancer agent.

Viral protein R (Vpr) is a 14kD, 96 amino acid accessory protein of the HIV virion that has been demonstrated to have important functions in the viral replication cycle including, among others, the induction of cell cycle arrest and apoptosis in rapidly proliferating cells, which results in immune dysfunction in infected individuals. Several investigators have studied the potential use of the apoptosis inducing and cell cycle arrest effect of Vpr as an anti-tumor therapeutic. In vitro studies have indicated that Vpr is cytotoxic against a large number of different tumor cell types including a number that are p53 independent. Likewise, some in vivo tumor studies using different delivery platforms/methods have indicated an anti-cancer effect mediated by Vpr. Our group has used the aggressive and poorly immunogenic murine melanoma tumor line B16.F10 as a model to deliver, through in vivo electroporation, Vpr expressing plasmids to established tumors and have demonstrated that this treatment regimen can induce growth attenuation and tumor regression in a proportion of the treated mice and appears to be associated with the induction of intratumoral apoptosis. Overall, to date, the data from a number of research groups, including our own, have indicated that Vpr has biological activity against a number of tumors in both in vivo and in vitro models and, as such, may be a potential candidate for testing in human clinical trials. In this report, we summarize the evidence supporting this hypothesis.

Anti-tumor activity mediated by protein and peptide transduction of HIV viral protein R (Vpr).

Peptides that are capable of traversing the cell membrane, via protein transduction domains (PTDs), are attractive either directly as drugs or indirectly as carriers for the delivery of therapeutic molecules. For example, an HIV-1 Tat derived peptide has successfully delivered a large variety of "cargoes" including proteins, peptides and nucleic acids into cells when conjugate to the PTD. There also exists other naturally occurring membrane permeable peptides which have potential as PTDs. Specifically, one of the accessory proteins of HIV (viral protein R; i.e., Vpr), which is important in controlling viral pathogenesis, possesses cell transduction domain characteristics. Related to these characteristics, Vpr has also been demonstrated to induce cell cycle arrest and host/target cell apoptosis, suggesting a potential anti-cancer activity for this protein. In this report we assessed the ability of Vpr protein or peptides, with or without conjugation to a PTD, to mediate anti-cancer activity against several tumor cell lines. Specifically, several Vpr peptides spanning carboxy amino acids 65-83 induced significant (i.e., greater than 50%) in vitro growth inhibition/toxicity of murine B16.F10 melanoma cells. Likewise, in in vitro experiments with other tumor cell lines, conjugation of Vpr to the Tat derived PTD and transfection of this construct into cells enhanced the induction of in vitro apoptosis by this protein when compared to the effects of transfection of cells with unconjugated Vpr. These results underscore the potential for Vpr based reagents as well as PTDs to enhance anti-tumor activity, and warrants further examination of Vpr protein and derived peptides as potential therapeutic agents against progressive cell proliferative diseases such as cancer.