Farnós Omar, Rodríguez Manuel, Chiong Maylin, Parra Francisco, Boué Oscar, Lorenzo Norailys, Colás Manuel, Lleonart Ricardo
Animal Biotechnology Division, Center for Genetic Engineering and Biotechnology, P.O. Box 6162, CP 10600, Havana, Cuba.
Vet Microbiol. 2006 May 31;114(3-4):187-95. doi: 10.1016/j.vetmic.2005.11.062. Epub 2005 Dec 27.
Rabbit hemorrhagic disease (RHD) is a contagious and highly lethal viral disease of rabbits that spreads rapidly and infects animals by nasal, conjunctival and oral routes. Therefore, this experiment was undertaken to study the immune response generated after intranasal (i.n.) vaccination with the recombinant VP60 capsid protein from rabbit hemorrhagic disease virus (RHDV) expressed at high levels in Pichia pastoris. Groups of BALB/c mice were immunized with three doses of purified VP60 protein (Group 1), VP60 formulated within the cell debris fraction of the transformed yeast (Group 2) and placebo (Group 3) by intranasal route. Mice were also intramuscularly injected with purified VP60 protein (Group 4). A rapid antibody response specific against rabbit hemorrhagic disease virus was observed in all the experimental groups, except in Group 3, as detected by ELISA. The highest titers were found 60 days after the first immunization. Mice from Group 1 showed the highest IgG response (p<0.05) and the most balanced profile of IgG1, IgG2a and IgG2b subclasses. IgA titers specific to the virus were found only in animals from this group, which also developed the highest specific lymphocyte proliferative response. Interferon-gamma (IFN-gamma) and interleukin-12 (IL-12) gene expression was also detected after an ex vivo-specific stimulation of mice from Groups 1 and 4. These data demonstrated the capacity of VP60 protein expressed in P. pastoris to elicit a potent humoral and cell-mediated immune response following an intranasal immunization scheme.
兔出血性疾病（RHD）是一种传染性极强且致死率高的兔病毒性疾病，传播迅速，可通过鼻腔、结膜和口腔途径感染动物。因此，本实验旨在研究用在毕赤酵母中高水平表达的兔出血性疾病病毒（RHDV）重组VP60衣壳蛋白经鼻内（i.n.）接种后产生的免疫反应。将BALB/c小鼠分为几组，通过鼻内途径分别用三剂纯化的VP60蛋白（第1组）、在转化酵母的细胞碎片组分中配制的VP60（第2组）和安慰剂（第3组）进行免疫。小鼠还通过肌肉注射纯化的VP60蛋白（第4组）。通过ELISA检测发现，除第3组外，所有实验组均观察到针对兔出血性疾病病毒的快速抗体反应。首次免疫后60天发现抗体滴度最高。第1组小鼠表现出最高的IgG反应（p<0.05）以及最平衡的IgG1、IgG2a和IgG2b亚类分布。仅在该组动物中发现了针对该病毒的特异性IgA滴度，并且该组动物还产生了最高的特异性淋巴细胞增殖反应。对第1组和第4组小鼠进行体外特异性刺激后，还检测到了干扰素-γ（IFN-γ）和白细胞介素-12（IL-12）基因表达。这些数据表明，毕赤酵母中表达的VP60蛋白在经鼻内免疫方案后能够引发强烈的体液免疫和细胞介导的免疫反应。
