Biophysical Studies of Liposome Encapsulated Pokeweed Antiviral Protein and its use as a HIV Therapeutic

Abstract

Human Immunodeficiency Virus (HIV) is a virus that attacks the human immune system, compromising its effect in regards to disease prevention. HIV results in the destruction of CD4 cells, which are vital in the defense against human immune responses. HIV can severely damage the immune system and lead to Acquired Immunodeficiency Syndrome (AIDS). There is presently no cure or effective HIV vaccine and as a result it is important to seek alternative measures in the defense against HIV/AIDS. Pokeweed Antiviral Protein (PAP), a protein isolated from pokeweed plant, Phytolacca americana, provides a new and profoundly promising direction in the field of HIV/AIDS research. PAP plays a vital role in the immune system of pokeweed and is a ribosome inactivating protein (RIP), inhibiting viral protein production. PAP possesses antiviral properties, and reduces the infectivity of many plant and animal viruses, including HIV-1. My project focuses on mechanisms using liposomal encapsulated PAP, targeted to infected cells as a therapeutic for HIV infection. Steady state fluorescence was used to identify the thermodynamic parameters of interaction between PAP and HIV RNA. PAP binds to the m7 GTP cap and the 5′ UTR region of HIV RNA. Various isoforms of PAP were tested and the optimal affinity was identified as a Km of 20.09 nM. Fluorescence observed V.S [m7 GTP] was plotted through nonlinear regression using the Prism 6.0 program. Varying liposomes were prepared in order to identify the optimal characteristics for PAP encapsulation. Liposomes were lyophilized through rotary evaporation and later extruded to ensure homogeneity. Fluorescent titration enables the testing of liposomal PAP encapsulation efficiency. It was concluded that a 1/1 v% mixture of liposomes DOPE-DOTAP is most efficient for PAP encapsulation. These accomplishments will enable further modification of the liposomes by covalently conjugating HIV-specific monoclonal antibodies (anti-pg120 and/or anti-pg41), thus facilitating the selectivity of the liposomal PAP to the target tissues (HIV-infected CD4+ lymphocytes). With the aforementioned completed the basis for PAP-liposome interactions can be established and also the modulation of these interactions in order to target HIV-infected cells.