Stevens Rosemary, Lavoy Alora, Nordone Shila, Burkhard Maryjo, Dean Gregg A
Department of Microbiology, Pathology and Parasitology, College of Veterinary Medicine, North Carolina State University, 4700 Hillsborough Street, Raleigh, NC 27606, USA.
Vaccine. 2005 Feb 10;23(12):1479-90. doi: 10.1016/j.vaccine.2004.09.033.
Listeria monocytogenes is an attractive biologic vaccine vector against HIV because it induces a strong cell mediated immune response, can be delivered by mucosal routes, can be readily manipulated to express viral antigens, and is easy and inexpensive to produce. Proof of concept studies have been performed using HIV Gag expressing recombinant L. monocytogenes in the mouse. Here we report the development and validation of recombinant L. monocytogenes to be evaluated in the FIV/cat model of HIV. Using a simplified approach to introduce individual and polyprotein FIV gag genes, we show that recombinant L. monocytogenes containing the entire gag expresses the full-length Gag polyprotein in a soluble secreted form. A DNA vaccine plasmid (pND14-Lc-env) that replicates in Gram positive bacteria and contains the FIV SU (gp100) and the ectodomain of TM (gp40) in a eukaryotic expression cassette was transfected into LM-gag to create LM-gag/pND14-Lc-env. After infection of target cells with LM-gag/pND14-Lc-env in vitro, both FIV Gag and Env proteins were detected in soluble cell lysates. Whether previous exposure to L. monocytogenes affects the immunogenicity of LM-gag/pND14-Lc-env was determined in cats infected with wild-type L. monocytogenes orally and/or subcutaneously. After a single oral dose of LM-gag/pND14-Lc-env, cats with existing anti-L. monocytogenes immune responses developed anti-FIV Gag IgA titers in vaginal secretions, saliva, and feces. Similarly, FIV Gag and Env specific IFN-gamma ELISPOT responses were measurable in spleen and lymph node but at a statistically higher frequency in cats exposed to a single subcutaneous dose of wild-type L. monocytogenes versus cats exposed both subcutaneously and orally. The FIV/cat model will provide a useful challenge system to determine whether recombinant L. monocytogenes can protect against a lentivirus in its natural host after challenge by the routes common to HIV transmission.
单核细胞增生李斯特菌是一种有吸引力的抗HIV生物疫苗载体，因为它能诱导强烈的细胞介导免疫反应，可以通过黏膜途径递送，能够很容易地进行改造以表达病毒抗原，并且生产简便、成本低廉。已经在小鼠中使用表达HIV Gag的重组单核细胞增生李斯特菌进行了概念验证研究。在此，我们报告了将在HIV的FIV/猫模型中进行评估的重组单核细胞增生李斯特菌的研发与验证情况。通过一种简化方法引入单个和多聚蛋白FIV gag基因，我们发现含有完整gag的重组单核细胞增生李斯特菌以可溶性分泌形式表达全长Gag多聚蛋白。将一种在革兰氏阳性细菌中复制且在真核表达盒中包含FIV SU（gp100）和TM胞外域（gp40）的DNA疫苗质粒（pND14-Lc-env）转染到LM-gag中，构建出LM-gag/pND14-Lc-env。在体外将LM-gag/pND14-Lc-env感染靶细胞后，在可溶性细胞裂解物中检测到了FIV Gag和Env蛋白。在经口服和/或皮下感染野生型单核细胞增生李斯特菌的猫中，确定了先前接触单核细胞增生李斯特菌是否会影响LM-gag/pND14-Lc-env的免疫原性。在单次口服一剂LM-gag/pND14-Lc-env后，具有现有的抗单核细胞增生李斯特菌免疫反应的猫在阴道分泌物、唾液和粪便中产生了抗FIV Gag IgA滴度。同样，在脾脏和淋巴结中可检测到FIV Gag和Env特异性的IFN-γ ELISPOT反应，但与经皮下和口服接触的猫相比，经单次皮下剂量接触野生型单核细胞增生李斯特菌的猫中该反应的频率在统计学上更高。FIV/猫模型将提供一个有用的挑战系统，以确定重组单核细胞增生李斯特菌在通过HIV传播常见途径进行攻击后，能否在其天然宿主中抵御慢病毒。
