Analysis of Cytotoxic T Lymphocyte Responses to SIV Proteins in SIV-Infected Macaques Using Antigen-Specific Stimulation with Recombinant Vaccinia and Fowl Poxviruses

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Abstract
Methods to analyze CD8+ CTL responses to simian immunodeficiency virus (SIV)-encoded proteins are essential to understand lentivirus immunopathogenesis and protective immune responses. Recombinant infectious shuttle vectors are useful for analyzing CTL responses to many viruses, including HIV. Therefore, CTL responses in SIV-infected Macaca fascicularis to SIV env and SIV gag/pol were evaluated using specific antigen stimulation with recombinant vaccinia (rVV) and fowl poxviruses (rFPV) containing SIV genes. Peripheral blood mononuclear cells from SIV-infected animals were stimulated with autologous cells infected with rVV expressing SIV env/gag/pol, and CTLs specific for SIV env and for SIV gag/pol were detected by testing for lytic activity in target cells expressing these genes separately. Lymphocyte subset purifications from the effector population demonstrated that the CTL response was mediated by CD8+ cells, and the use of brefeldin A to selectively block antigen presentation in association with MHC class I products affirmed this cytolytic activity was class I restricted. The use of rVV to analyze responses to SIV genes is potentially problematic in hosts immunized to vaccinia. Fowl poxvirus is an alternative virus that has many of the molecular advantages of vaccinia virus but is genomically distinct. Therefore, the ability of rFPV to expand and detect SIV-specific CTLs was evaluated. Although there was no cytopathic effect following infection with rFPV, macaque cells infected with this vector did express rFPV gene products, and could be used as stimulator and target cells to detect SIV-specific CD8+ CTLs. The results suggest that these recombinant viral vectors can be used to specifically stimulate CD8+, MHC class I-restricted CTLs reactive to SIV proteins, and should facilitate evaluating CTL responses in both SIV-infected animals and animals vaccinated against SIV.

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