Bounds Callie E, Kwilas Steven A, Kuehne Ana I, Brannan Jennifer M, Bakken Russell R, Dye John M, Hooper Jay W, Dupuy Lesley C, Ellefsen Barry, Hannaman Drew, Wu Hua, Jiao Jin-an, Sullivan Eddie J, Schmaljohn Connie S
United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Maryland, United States of America.
Ichor Medical Systems, Inc., San Diego, California, United States of America.
PLoS One. 2015 Sep 30;10(9):e0137786. doi: 10.1371/journal.pone.0137786. eCollection 2015.
DNA vaccination of transchromosomal bovines (TcBs) with DNA vaccines expressing the codon-optimized (co) glycoprotein (GP) genes of Ebola virus (EBOV) and Sudan virus (SUDV) produce fully human polyclonal antibodies (pAbs) that recognize both viruses and demonstrate robust neutralizing activity. Each TcB was vaccinated by intramuscular electroporation (IM-EP) a total of four times and at each administration received 10 mg of the EBOV-GPco DNA vaccine and 10 mg of the SUDV-GPco DNA vaccine at two sites on the left and right sides, respectively. After two vaccinations, robust antibody responses (titers > 1000) were detected by ELISA against whole irradiated EBOV or SUDV and recombinant EBOV-GP or SUDV-GP (rGP) antigens, with higher titers observed for the rGP antigens. Strong, virus neutralizing antibody responses (titers >1000) were detected after three vaccinations when measured by vesicular stomatitis virus-based pseudovirion neutralization assay (PsVNA). Maximal neutralizing antibody responses were identified by traditional plaque reduction neutralization tests (PRNT) after four vaccinations. Neutralizing activity of human immunoglobulins (IgG) purified from TcB plasma collected after three vaccinations and injected intraperitoneally (IP) into mice at a 100 mg/kg dose was detected in the serum by PsVNA up to 14 days after administration. Passive transfer by IP injection of the purified IgG (100 mg/kg) to groups of BALB/c mice one day after IP challenge with mouse adapted (ma) EBOV resulted in 80% protection while all mice treated with non-specific pAbs succumbed. Similarly, interferon receptor 1 knockout (IFNAR(-/-)) mice receiving the purified IgG (100 mg/kg) by IP injection one day after IP challenge with wild type SUDV resulted in 89% survival. These results are the first to demonstrate that filovirus GP DNA vaccines administered to TcBs by IM-EP can elicit neutralizing antibodies that provide post-exposure protection. Additionally, these data describe production of fully human IgG in a large animal system, a system which is capable of producing large quantities of a clinical grade therapeutic product.
用表达埃博拉病毒（EBOV）和苏丹病毒（SUDV）密码子优化（co）糖蛋白（GP）基因的DNA疫苗对转染色体牛（TcB）进行DNA疫苗接种，可产生完全人源多克隆抗体（pAb），该抗体可识别两种病毒并表现出强大的中和活性。每头TcB通过肌肉内电穿孔（IM-EP）总共接种四次，每次接种时分别在左右两侧的两个部位接受10mg的EBOV-GPco DNA疫苗和10mg的SUDV-GPco DNA疫苗。两次接种后，通过ELISA检测到针对全辐照EBOV或SUDV以及重组EBOV-GP或SUDV-GP（rGP）抗原的强大抗体反应（效价>1000），rGP抗原的效价更高。通过基于水疱性口炎病毒的假病毒中和试验（PsVNA）测量，三次接种后检测到强烈的病毒中和抗体反应（效价>1000）。四次接种后通过传统的蚀斑减少中和试验（PRNT）确定了最大中和抗体反应。从三次接种后收集的TcB血浆中纯化的人免疫球蛋白（IgG），以100mg/kg的剂量腹腔内（IP）注射到小鼠体内，在给药后长达14天通过PsVNA在血清中检测到中和活性。在用小鼠适应株（ma）EBOV进行IP攻击一天后，将纯化的IgG（100mg/kg）通过IP注射被动转移到BALB/c小鼠组中，可提供80%的保护，而所有用非特异性pAb治疗的小鼠均死亡。同样，在用野生型SUDV进行IP攻击一天后，通过IP注射接受纯化IgG（100mg/kg）的干扰素受体1基因敲除（IFNAR（-/-））小鼠的存活率为89%。这些结果首次证明，通过IM-EP给TcB接种丝状病毒GP DNA疫苗可引发中和抗体，提供暴露后保护。此外，这些数据描述了在大型动物系统中完全人源IgG的产生，该系统能够生产大量临床级治疗产品。
