Rodriguez F, Zhang J, Whitton J L
Department of Neuropharmacology, The Scripps Research Institute, La Jolla, California 92037, USA.
J Virol. 1997 Nov;71(11):8497-503. doi: 10.1128/JVI.71.11.8497-8503.1997.
DNA immunization can induce cytotoxic T lymphocytes (CTL), antibodies, and protection against microbial challenge. The underlying mechanisms remain obscure and must be understood to permit rational manipulation and optimization of the technique. We set out to enhance the intracellular degradation of a viral antigen, with the intent of improving antigen entry into, and presentation by, the class I major histocompatibility complex pathway. We achieved this goal by cotranslational ubiquitination of a plasmid-encoded viral antigen, lymphocytic choriomeningitis virus (LCMV) nucleoprotein (NP). We show that native NP is very stable in cell culture, while the ubiquitinated product is so rapidly degraded that it is barely detectable. This rapid degradation leads to more efficient sensitization of target cells in an in vitro cytotoxicity assay, consistent with enhanced antigen presentation, and both degradation and target cell recognition are blocked by a proteasome inhibitor. We have used the plasmid for in vivo studies and find that, remarkably, ubiquitination leads to a complete abrogation of antibody responses, presumably because the encoded protein is so rapidly and completely degraded that insufficient antigen remains to interact appropriately with B cells. In contrast, in vivo CTL induction is improved by ubiquitination of NP. That CTL are induced at all by this rapidly degraded protein may shed light on the mechanism by which CTL are induced by DNA immunization; it has been suggested that CTL induction following intramuscular DNA injection results not from antigen presentation by cells taking up and expressing the DNA but rather from uptake of soluble protein by specialized antigen-presenting cells (APC). It appears to us unlikely that the ubiquitinated protein could function in this manner, since it is so rapidly degraded in vitro and fails to induce antibodies in vivo. Finally, the ubiquitinated protein confers markedly enhanced protection against LCMV challenge. Mice immunized with a plasmid encoding NP show approximately 100-fold reductions in virus titers compared to controls, while mice immunized with a plasmid encoding the ubiquitinated NP show reductions in virus load of at least 5 x 10(4)- to 5 x 10(5)-fold. This is by far the most effective DNA vaccine that we have yet designed. Ubiquitination therefore may improve DNA immunization, but caution is warranted, since immunity to many microbes depends on induction of good humoral immunity, and we show here that this may be prevented by ubiquitination of the encoded protein.
DNA免疫可诱导细胞毒性T淋巴细胞（CTL）、抗体产生，并提供针对微生物攻击的保护作用。其潜在机制仍不清楚，必须加以了解才能合理地操控和优化该技术。我们着手增强病毒抗原的细胞内降解，目的是改善抗原进入I类主要组织相容性复合体途径并由其呈递的情况。我们通过对质粒编码的病毒抗原——淋巴细胞性脉络丛脑膜炎病毒（LCMV）核蛋白（NP）进行共翻译泛素化来实现这一目标。我们发现天然NP在细胞培养中非常稳定，而泛素化产物降解极快，几乎检测不到。这种快速降解导致在体外细胞毒性试验中靶细胞的致敏更有效，这与增强的抗原呈递一致，并且降解和靶细胞识别均被蛋白酶体抑制剂阻断。我们已将该质粒用于体内研究，并且发现，值得注意的是，泛素化导致抗体反应完全消除，推测是因为编码的蛋白质降解得如此迅速和彻底，以至于没有足够的抗原残留来与B细胞进行适当相互作用。相比之下，NP的泛素化可改善体内CTL的诱导。这种快速降解的蛋白质居然能诱导CTL，这可能会揭示DNA免疫诱导CTL的机制；有人提出，肌内注射DNA后CTL的诱导并非源于摄取并表达DNA的细胞进行的抗原呈递，而是源于专门的抗原呈递细胞（APC）对可溶性蛋白质的摄取。在我们看来，泛素化蛋白不太可能以这种方式发挥作用，因为它在体外降解极快且在体内无法诱导抗体产生。最后，泛素化蛋白赋予对LCMV攻击的显著增强的保护作用。与对照相比，用编码NP的质粒免疫的小鼠病毒滴度降低约100倍，而用编码泛素化NP的质粒免疫的小鼠病毒载量降低至少5×10⁴至5×10⁵倍。这是我们迄今设计的最有效的DNA疫苗。因此，泛素化可能会改善DNA免疫，但需谨慎，因为对许多微生物的免疫依赖于良好的体液免疫的诱导，而我们在此表明编码蛋白的泛素化可能会阻止这种情况发生。
