Infection with the human immunodeficiency virus (HIV) remains a formidable health problem worldwide. Development of effective antiviral agents is central to the control of HIV infection and prevention of AIDS. In the search for novel anti-HIV agents, secretory leukocyte protease inhibitor (SLPI), was recently identified as an endogenous inhibitor of HIV-l. Naturally present in serous secretions such as saliva, this 12 kDa serine protease- specific inhibitor potently blocks HIV-l infectivity of mononuclear cells at physiologic concentrations (1-10 mu g/ml). A logical extension of this finding is to determine whether enhanced SLPI expression will protect cells against infection in vivo. This knowledge will provide insights as to the potential use of SLPI as an HIV therapeutic and contribute to the knowledge of HIV biology. As a first step to evaluate the feasibility of using SLPI-mediated gene therapy for HIV, the proposed preliminary study will test that hypothesis that SLPI-secreting cells are protected from infection with HIV-l. Toward this end, a model using retroviral vector- mediated gene transfer will be developed in which primary peripheral blood lymphocytes (PBL, naturally susceptible to viral infection) are genetically engineered to express soluble SLPI. The -SLPI-secreting cells will be tested for the ability to resist HIV- 1 infection.
In specific aim 1, a selectable retroviral vector containing human SLPI cDNA will be generated.
In specific aim 2, SLPI-enconding vector DNA will be delivered to PBL as follows. Amphotropic producer cells will be transiently transfected with vector DNA. Retrovirus-containing supernatants will be harvested, titered and used to infect (transduce) activated PBL. Vector- containing cells will be selected using puromycin and characterized for cellular phenotype and toxicity. Cellular integration of the vector will be confirmed by PcR analysis. Soluble SLPI -expression will be quantitated using an enzyme-linked immunosorbent assay (ELISA).
In specific aim 3, the SLPI-secreting cells will be challenged with HIV-l in an infectivity assay. Infectivity will be quantitated by viral p24 antigen ELISA and compared with soluble SLPI levels. The model will be optimized using the HIV-1 11113 strain and then extended to evaluate infectivity using other HIV-l strains (MN, RF, NL4-3) and clinical (clinical or field) HIV-1 isolates. The proposed preliminary study will form the basis of a future R0l NIH grant application to more fully explore this exciting and novel approach in the fight against AIDS.
| Shugars, D C; Alexander, A L; Fu, K et al. (1999) Endogenous salivary inhibitors of human immunodeficiency virus. Arch Oral Biol 44:445-53 |
