Choi A H, Basu M, Rae M N, McNeal M M, Ward R L
Division of Infectious Diseases, Children's Hospital Medical Center, Cincinnati, Ohio, 45229-3039, USA.
Virology. 1998 Oct 10;250(1):230-40. doi: 10.1006/viro.1998.9370.
We recently reported that epidermal immunization using the PowderJet particle delivery device with plasmid vector pcDNA1/EDIM6 encoding rotavirus VP6 of murine strain EDIM induced high levels of serum rotavirus IgG but failed to protect mice against EDIM infection (Choi, A. H., Knowlton, D. R., McNeal, M. M., and Ward, R. L. (1997) Virology 232, 129-138.). This was extended to determine whether pcDNA1/EDIM4 or pcDNA1/EDIM7, which encode either rotavirus VP4 or VP7, the rotavirus neutralization proteins, could also induce rotavirus-specific antibody responses and if these responses resulted in protection. Titers of rotavirus serum IgG increased with the first dose in mice immunized with pcDNA1/EDIM7, but little or no serum rotavirus IgG was detected in mice immunized with pcDNA1/EDIM4. In vitro assays with these plasmids in rabbit reticulocyte lysates showed that VP4 was expressed but the amount was considerably lower than VP6 or VP7. To improve expression of VP4 and induction of rotavirus-specific humoral responses, the coding region of VP4 was cloned into the high-expression plasmid WRG7054 as a fusion protein containing the 22-amino-acid secretory signal peptide of tissue plasminogen activator (tPA) at its N terminus. In vitro expression of tPA::VP4 was significantly higher than unmodified VP4, and mice inoculated with WRG7054/EDIM4 generated high titers of rotavirus IgG. The coding sequence of VP7 without the first 162 nucleotides was also cloned into WRG7054, but no difference was observed between titers of serum rotavirus IgG in mice immunized with this plasmid (WRG7054/EDIM7Delta1-162) and pcDNA1/EDIM7. The rotavirus-specific IgG titers in all immune sera were predominantly IgG1 indicating induction of Th 2-type responses. None of the mice immunized with any of the VP4 or VP7 plasmids developed serum or fecal rotavirus IgA or neutralizing antibody to EDIM. When immunized mice were challenged with EDIM virus, there was no significant reduction in viral shedding relative to unimmunized controls. Therefore epidermal immunization with VP4 or VP7 alone elicited rotavirus IgG responses but did not protect against homologous rotavirus challenge.
我们最近报道，使用PowderJet颗粒递送装置进行表皮免疫，用编码鼠源EDIM株轮状病毒VP6的质粒载体pcDNA1/EDIM6可诱导高水平的血清轮状病毒IgG，但不能保护小鼠免受EDIM感染（Choi, A. H., Knowlton, D. R., McNeal, M. M., and Ward, R. L. (1997) Virology 232, 129 - 138.）。在此基础上进行扩展研究，以确定编码轮状病毒中和蛋白VP4或VP7的pcDNA1/EDIM4或pcDNA1/EDIM7是否也能诱导轮状病毒特异性抗体反应，以及这些反应是否能提供保护。在用pcDNA1/EDIM7免疫的小鼠中，轮状病毒血清IgG滴度随首剂增加，但在用pcDNA1/EDIM4免疫的小鼠中，几乎检测不到血清轮状病毒IgG或检测到的量很少。在兔网织红细胞裂解物中对这些质粒进行的体外试验表明，VP4有表达，但表达量明显低于VP6或VP7。为了提高VP4的表达并诱导轮状病毒特异性体液反应，将VP4的编码区克隆到高表达质粒WRG7054中，作为一种融合蛋白，其N端含有组织型纤溶酶原激活剂（tPA）的22个氨基酸的分泌信号肽。tPA::VP4的体外表达明显高于未修饰的VP4，接种WRG7054/EDIM4的小鼠产生了高滴度的轮状病毒IgG。VP7的编码序列去除前162个核苷酸后也被克隆到WRG7054中，但用该质粒（WRG7054/EDIM7Delta1 - 162）免疫的小鼠与用pcDNA1/EDIM7免疫的小鼠相比，血清轮状病毒IgG滴度没有差异。所有免疫血清中的轮状病毒特异性IgG滴度主要为IgG1，表明诱导了Th2型反应。用任何VP4或VP7质粒免疫的小鼠均未产生血清或粪便轮状病毒IgA或针对EDIM的中和抗体。当用EDIM病毒攻击免疫小鼠时，相对于未免疫的对照，病毒排出量没有显著减少。因此，单独用VP4或VP7进行表皮免疫可引发轮状病毒IgG反应，但不能保护小鼠免受同源轮状病毒攻击。
