摘要
目的以人类免疫缺陷病毒1型（HIV-1）疫苗免疫BALB／c（H-2^d）小鼠为模型，探讨有效活化黏膜与系统免疫力的策略。方法将6～8周龄BALB／c小鼠随机分为6组，每组3只；利用DNA疫苗加或不加疫苗佐剂滴鼻途径进行黏膜引导免疫，利用重组痘苗天坛株疫苗进行系统加强的策略以提高机体免疫反应。DNA疫苗免疫时间是0、14、28d，剂量每只小鼠10μg/次；重组痘苗病毒加强免疫时间是第42天，剂量每只小鼠1×10^7PFU／次。初次免疫后第56天处死小鼠。ELISPOT和胞内染色测定T淋巴细胞免疫反应；ELISA法测血清特异的IgG和鼻咽部、肺部灌洗液特异的IgA。结果单独DNA疫苗黏膜免疫后只活化微弱的系统T细胞免疫（2±1）斑点形成细胞（spotformingcells．SFC／10^6个细胞），黏膜共同施用霍乱毒素可提高相应的系统T细胞免疫力（14±11SFC／10^6个细胞），重组痘苗天坛株疫苗系统加强后，分泌γ-干扰素（IFN-γ）的特异性T淋巴细胞升高4．5倍（61±35 SFC／10^6个细胞）；胞内染色可获得1．8％±1．4％HIV-1 Gag特异性CD8^＋T细胞免疫反应，同时，血清中特异性IgG抗体与肺部灌洗液的特异性IgA抗体水平在痘苗天坛株疫苗系统加强后分别提高2倍[吸光度（A）值：1．5±0．3]和2．5倍（A值：1．8±0．8）。结论黏膜进行引导免疫并辅以系统加强的免疫策略可诱发有效的黏膜、系统体液免疫反应和T淋巴细胞免疫。
Objective To explore the strategy to raise both mucosal and systemic anti-HIV-1 immunity. Methods Eighteen BALB/c rats were randomly divided into 2 groups, experimental group and control group. The experimental group were further subdivided into 4 subgroups of 3 mice： 3-dose HIV DNA vaccine group, 3-dose DNA vaccine ± cholera toxin （CT） adjuvant subgroup, 1-dose recombinant Tiantan strain vaccinia-based vaccine subgroup, and 3-dose DNA vaccine ＋ CT adjuvant ＋ Tiantan strain vacciniabased vaccine subgroup. The control group was subdivided into 2 subgroups of 3 mice ： 3-dose DNA blank vector subgroup, and 3-dose DNA blank vector ＋ Tiantan strain vaccinia-based vaccine subgroup. Intranasal administration of DNA vaccine-based vaccine （ 10 p.g） was done on the days 0, 14, and 28 as the mucosal priming, and recombinant Tiantan vaccinia （ 1 ×10^7 PFU） was injected intramuscularly as systemic boosting on the day 42. On the day 56 the mice were killed and specimens of serum, nasopharynx wash, lung wash, and spleen were collected and splenocytes were isolated. Splenocytes were added into the phosphate-buffered saline with anti-mouse interferon-γ （IFN-γ） envelop antibody to count the number of spot-forming cells （SFCs）. Indirect ELISA was used to detect the HIV-1 specific antibody in the nasopharynx wash and lung wash. Immunohistochemistry was used to detect the intracellular staining of IFN-γ in the splenocytes. Results The number of spot forming cells in the HIV-1 DNA vaccine ＋ CT adjuvant group was （ 14 ± 11 ） SFCs/10^6 splenocytes, significantly more than that of the HIV-1 DNA vaccine group [ （ 2 ± 1 ） SFCs/106 spleen cells （P 〈0.01 ）. The number of SFCs of the 1-dose DNA-vaccine subgroup was [ （30 ± 18） SFCs/10^6 spleen cells ], significantly higher than that of the only DNS vaccine group （ P 〈 0.01 ）. The number of SFCs of DNA vaccine ＋ CT adjuvant ＋ recombinant Tiantan vacciniabased vaccine was （61 ± 35 ）SFCs/10^6 splenocytes, significantly higher than those of the other groups （all P 〈0.01 ）. Flow cytometry showed that the rate of HIV-1 Gag specific CD8^＋ T cell was 1.8% -± 1.4%. The value of specific IgG of the DNA vaccine ＋ adjuvant ＋ Tiantan vaccinia- based vaccine was 1.50 ± 0.30, significantly higher than those of the blank vector, single-dose Tiantan vaccinia- based vaccine, and single-dose DNA vaccine ＋ CT adjuvant subgroups （0.42±0.02, 0.74 ± 0. 13, and 0.75 ± 0. 02 respectively, all P 〈 0.05）. In different subgroups the levels of specific IgA in the lung wash were all higher than those in the nasopharynx wash. The levels of specific IgA in the lung and nasopharynx wash of the DNA vaccine ＋ CT adjuvant subgroup were higher than those of the other subgroups whether or not with boosting of Tiantan. The specific IgA levels of the groups enhanced by Tiantan vacciniabased vaccine were all significantly higher than those of the corresponding subgroups without enhancement （ all P 〈 0.01 ）. The IgA level of lung wash of the DNA vaccine ＋ CT adjuvant subgroup was 1.82 ± 0.76, significantly higher than that of the one-dose Tiantan vacciniabased vaccine group （0.52 ± 0.19, P 〈 0.05 ）. Conclusion The vaccination modality of mucosal priming and systemic boosting induces both mucosal and systemic immune responses.
出处
《中华医学杂志》
CAS
CSCD
北大核心
2006年第44期3109-3113,共5页
National Medical Journal of China
基金
国家科技部"973"资助项目(2005CB522903)
中国综合性艾滋病研究项目资助(U19AIS1915-03)
