Cutting Edge: Selective role of ubiquitin in MHC class I antigen presentation.

The importance of ubiquitination in MHC class I-restricted Ag processing remains unclear. To address this issue, we overexpressed wild-type and dominant-negative lysineless forms of ubiquitin (Ub) in mammalian cells using an inducible vaccinia virus system. Overexpression of the lysineless Ub nearly abrogated polyubiquitination and potently inhibited epitope presentation from a cytosolic N-end rule substrate as well as endoplasmic reticulum (ER)-targeted model Ags. In contrast, there was little impact on Ag presentation from cytosolic proteins. These trends were location dependent; redirecting cytosolic Ag to the ER rendered presentation lysineless Ub-sensitive, whereas retargeting exocytic Ag to the cytosol had the inverse effect. This dichotomy was further underscored by small interfering RNA knockdown of the ER-associated Ub ligase Hrd1. Thus, Ub-dependent degradation appears to play a major role in the MHC class I-restricted processing of ER-targeted proteins and a more restricted role in the processing of cytosolic proteins.

Enhanced humoral HIV-1-specific immune responses generated from recombinant rhabdoviral-based vaccine vectors co-expressing HIV-1 proteins and IL-2.

Recombinant rabies virus (RV) vaccine strain-based vectors expressing HIV-1 antigens have been shown to induce strong and long-lasting cellular but modest humoral responses against the expressed antigens in mice. However, an effective vaccine against HIV-1 may require stronger responses, and the development of such an immune response may depend on the presence of certain cytokines at the time of the inoculation. Here, we describe several new RV-based vaccine vehicles expressing HIV-1 Gag or envelope (Env) and murine IL-2 or IL-4. Cells infected with recombinant RVs expressed high levels of functional IL-2 or IL-4 in culture supernatants in addition to HIV-1 proteins. The recombinant RV expressing IL-4 was highly attenuated in a cytokine-independent manner, indicating that the insertion of two foreign genes into the RV genome is mainly responsible for the attenuation observed. The expression of IL-4 resulted in a decrease in the cellular immune response against HIV-1 Gag and Env when compared with the parental virus not expressing IL-4 and only 2 of 20 mice seroconverted to HIV-1 Env after two inoculations. The IL-2-expressing RV was completely apathogenic after direct intracranial inoculation of mice. In addition, mice immunized with IL-2 maintained strong anti-HIV-1 Gag and Env cellular responses and consistently induced seroconversion against HIV-1 Env after two inoculations. This suggests the potential use of IL-2 in RV-based HIV-1 vaccine strategies, which may require the induction of both arms of the immune response.