Abstract
Despite tremendous progress in our understanding of human immunodeficiency virus (HIV) natural history and advances in HIV treatment, there is neither an approved vaccine nor a cure for infection. Here, we describe the development and characterization of a novel replicating vaccine vector utilizing Cytomegalovirus (CMV) and a TLR5 adjuvant. After partial truncation of the central, immunodominant hypervariable domain, flagellin (fliC) from Salmonella was cloned downstream of a codon optimized gag gene from simian immunodeficiency virus (SIV) and transiently expressed in telomerized rhesus fibroblast (TeloRF) cells in culture. Lysates generated from these transfected cells induced the tumor necrosis factor alpha (TNF-α), in a mouse macrophage cell line, in a TLR5-dependent manner. The Gag/FliC expression construct was cloned into a bacterial artificial chromosome encoding the rhesus CMV (RhCMV) genome, and infectious RhCMV was generated following transfection of TeloRF cells. This virus stably expressed an SIV Gag/FliC fusion protein through four serial passages. Lysates generated from infected cells induced TNF-α in a TLR5-dependent manner. Western blot analysis of infected cell lysates verified expression of a Gag/FliC fusion protein using a SIV p27 capsid monoclonal antibody. Lastly, rhesus macaques inoculated with this novel RhCMV virus demonstrated increased inflammatory responses at the site of inoculation seven days post-infection when compared to the parental RhCMV. These results demonstrate that an artificially constructed replicating RhCMV expressing an SIV Gag/FliC fusion protein is capable of activating TLR5 in a macrophage cell line in vitro and induction of an altered inflammatory response in vivo. Ongoing animals studies are aimed at determining vaccine efficacy, including subsequent challenge with pathogenic SIV.
Highlights
Thirty years of research has revealed major insights into the human immunodeficiency virus (HIV-1) replication cycle, pathogenesis, and immune responses; there is neither an approved vaccine nor a broadly applicable cure for HIV-1 infection
The results demonstrated that RhCMVgagfliCΔ196–378 induced production of tumor necrosis factor-α (TNF-α) in a Toll-like receptor 5 (TLR5)-dependent manner and that this activation of TLR5 was stable through all four serial passages
RhCMVgagfliCΔ196-378-infected telomerized rhesus fibroblast (TeloRF) cell lysates contained the 57 kD simian immunodeficiency virus (SIV) Gag protein, the unknown 40 kD protein, and another band at the expected 93 kD weight of the SIV Gag-FliCΔ196–378 fusion protein (854 amino acids). All three of these proteins (40 kD, 57 kD, and 93 kD) increased in concentration in the infected cells over time, peaking in concentration at approximately 72 hours post-infection. These results demonstrated that RhCMVgagfliCΔ196–378 in TeloRF cells expressed a fusion protein that cross-reacted with an SIV Gag (p27) monoclonal antibody at the appropriate size for a full-length SIV Gag-FliCΔ196–378 fusion protein
Summary
Thirty years of research has revealed major insights into the human immunodeficiency virus (HIV-1) replication cycle, pathogenesis, and immune responses; there is neither an approved vaccine nor a broadly applicable cure for HIV-1 infection. Combination antiretroviral therapy (cART) [1] is capable of controlling HIV-1 infection, low-level viremia persists [2] and virus in plasma rebounds if cART is interrupted [3]. HIV-1 infected people must remain on cART indefinitely to prevent further destruction of their immune systems and progression to acquired immune deficiency syndrome (AIDS). Non-AIDS associated diseases in people undergoing long-term treatment demonstrate that cART does not completely restore health and emphasize the need for a vaccine or a cure (reviewed in [4, 5])
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