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122 Chemically Inactivated HIV/SIV Virions with Functional Envelope Glycoproteins: In Vitro Applications
J. L. Rossio*1, M. Piatak, Jr.1, I. Frank2, M. Larsson2, N. Romani3, L. Henderson1, L. Arthur1, N. Bhardwaj2, M. Pope2, and J. D. Lifson1
1AVP, SAIC Frederick, NCI at Frederick, MD, USA; 2Rockefeller Univ., New York, NY, USA; and 3Univ. of Innsbruck, Austria
Background: We used a novel chemical inactivation process to inactivate the infectivity of HIV and SIV virions, while preserving the conformational and functional integrity of virion surface proteins, including the viral envelope glycoproteins. We have evaluated these particles in various in vitro applications.
Methods: We performed standard lymphoproliferation (LPA) and ELISPOT assays on whole PBMC or CD4- or CD8-depleted human or rhesus macaque PBMC populations, using inactivated virions as the stimulating antigen. We also studied the uptake of inactivated virions by dendritic cells and the ability of inactivated virions to be "cross-presented" by DC to stimulate MHC-I restricted responses by CD8+ T cells.
Results: Inactivated HIV and SIV virions stimulated virus-specific LPA and ELISPOT responses with comparable or greater sensitivity relative to available peptide or recombinant protein antigens. Lymphoproliferative responses were seen in both CD4+ and CD8+ populations. Inactivated SIV virions were taken up by both macaque and human DCs comparably to native infectious virions, apparently by clathrin-coated pits, with greater uptake by immature DCs compared with in vitro matured DCs. Virions with heat-denatured envelope glycoproteins were taken up much less efficiently. In mature DCs, large numbers of intact virions localized to intracytoplasmic vacuoles, often in perinuclear locations; fewer intact virions were seen in more peripheral areas in immature DCs. DCs pulsed with either inactivated HIV virions or apoptotic cells previously exposed to inactivated virions effectively presented antigen to stimulate virus-specific responses by CD8+ cells.
Conclusions: Chemically inactivated HIV and SIV virions with functional envelope glycoproteins represent a simple, versatile approach to studying virus/target cell interactions in the absence of confounding effects due to productive viral infection. They also appear to be a useful reagent for the characterization of virus-specific cellular immune responses in vitro.
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