Intracellular CytofIuorometric Detection of Productive HIV Infection: a TooI for Studying HIV Pathogenesis

Abstract

Intensive research conducted during the last 20 years has allowed the human immunodeficiency virus (HIV) to become one of the most characterized viruses known to mankind. Throughout the developed world, millions of lives have been spared through the efforts of prevention and treatment. Treatment in the form of anti-retroviral drugs or highly active antiretroviral therapy (HAART) has improved and prolonged the lives of millions of HIV-infected individuals and reduced the incidence of acquired immunodeficiency syndrome (AIDS). However, with all our knowledge and efforts we still have no cure, no vaccine and a treatment that is only available to a fraction of those affected by HIV. Even with current treatment regimens, eradication of the virus from infected individuals no longer seems realistic. The focus of treatment has now been placed on the immune system, in controlling the virus and preventing disease. As such, it becomes increasingly important to gain a better and clearer understanding of the pathogenesis associated with HIV infection, so that strategic immune-reconstituting therapies can be developed. Immune cell abnormalities that occur following HIV infection are a prime indication of viral pathogenesis, though most of the cellular abnormalities described to date have not been directly correlated with viral replication within the affected cell. In essence, it is not known whether the pathogenic abnormalities observed are directly or indirectly associated with productive HIV infection. To address this issue, a cytofluorometric assay for detecting productively HIV-infected cells was developed using broadly reactive HIV core protein specific antibodies. The assay was optimized and assessed for its ability to detect heterologous HIV-1 strain core proteins within infected cells and applied to studying pathogenesis by simultaneously measuring changes in cell surface and intracellular proteins in in vitro infected cell lines and primary cells. Additionally, the usefulness of the assay in antiviral therapy research as well as in detecting productively in vivo infected lymphocytes from HIV-infected donors was assessed. Overall, the optimized intracellular HIV detection assay proved to be highly specific and relatively sensitive for detecting intracellular core protein, and exhibited a cellular detection limit of 1 productively infected cell per 24,450 total cells. The assay also proved useful for associating phenotypic changes with cellular HIV infection by allowing the infected populations to be divided into corresponding productively and non-productively infected subpopulations. Separate analysis of these subpopulations allowed for changes in surface markers and intracellular cytokines to be unmasked as well as cellular changes previously associated with HIV infection to be dissected with respect to productive HIV infection, giving insight into the direct and indirect viral pathogenic mechanisms responsible.