INDUCTION OF MHC CLASS I-RESTRICTED CTL RESPONSE BY DNA IMMUNIZATION WITH UBIQUITIN-INFLUENZA VIRUS NUCLEOPROTEIN FUSION ANTIGENS

DNA vaccines have been shown to be an effective means of inducing cyto toxic T-lymphocyte (CTL) responses in both young and aged mice. Better understanding of the pathways by which antigens encoded by DNA vaccin es are processed and presented to CTL may allow for improvements in CT L responses in older animals. Since CTL recognize short peptides prese nted by MHC class I molecules, and since ubiquitin-dependent proteolys is is widely believed to be responsible for degradation of endogenousl y synthesized antigens and generation of these peptide ligands, we sou ght to use ubiquitin (Ub) conjugation to target influenza virus nucleo protein (NP) antigen into the Ub-proteasome degradation pathway for MH C class I-restricted antigen processing and presentation. However; the addition of the Ub moiety did not affect the half-life of Ub-NP prote in in transiently transfected human rhabdomyosarcoma (RD) cells. Moreo ver the modifications of NP DNA vaccine with Ub conjugation did not af fect their ability to induce a CTL response specific for the H-2K(d)-r estricted NP147-155 epitope, as assessed bq, both percent cytolysis in bulk CTL culture and by CTL precursor (CTLp) frequency in limiting di lution analysis (LDA). In contrast, the anti-NP antibody (Ab) response s were dramatically reduced in mice immunized with low doses (1 mu g) of Ub-NP constructs, compared with mice immunized with wild-type NP DN A, These results demonstrate that Ub conjugation alone does not guaran tee targeting of endogenously synthesized antigens for rapid degradati on by proteasomes. Furthermore, the ability of ubiquitination to reduc e Ab responses to NP without affecting CTL responses suggests that the Ub modifications result in a lower availability of full-length NP fro m transfected cells in vivo. The implications of these data on antigen presentation and cross-priming are discussed. (C) 1998 Elsevier Scien ce Ltd. All rights reserved.