Biesova Zuzana, Miller Mark A, Schneerson Rachel, Shiloach Joseph, Green Kim Y, Robbins John B, Keith Jerry M
Program in Developmental and Molecular Immunity, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892-2423, United States.
Vaccine. 2009 Oct 19;27(44):6234-8. doi: 10.1016/j.vaccine.2009.07.107. Epub 2009 Aug 15.
Production of influenza vaccines requires a minimum of 6 months after the circulating strain is isolated and the use of infectious viruses. The hemagglutinin (protective antigen) of circulating influenza viruses mutates rapidly requiring reformulation of the vaccines. Our goal is to eliminate the risk of working with infectious virus and reduce significantly the production time. A cDNA fragment encoding the influenza virus A/Vietnam/1203/2004 (H5N1) HA gene was prepared using RT-PCR with viral RNA as a template. Recombinant HA (rHA) protein was produced in Escherichia coli and purified from isolated inclusion bodies by urea solubilization and Ni(+)-ion column chromatography. Vaccine candidates were prepared by treating the rHA with formalin, adsorption onto alum or with both. Mice were injected subcutaneously with candidate vaccines two or three times 2 weeks apart. Sera were collected 1 week after the last injection and antibody measured by ELISA and hemagglutination inhibition (HI). The highest antibody response (GM 449EU) was elicited by three injections of 15microg alum-adsorbed rHA. Dosages of 5microg of rHA formulated with formalin and alum, and 5microg alum-adsorbed rHA elicited IgG anti-HA of GM 212 and 177EU, respectively. HI titers, >or=40 were obtained in >or=80% of mice with three doses of all formulations. We developed a method to produce rHA in a time-frame suitable for annual and pandemic influenza vaccination. Using this method, rHA vaccine can be produced in 3-4 weeks and when formulated with alum, induces HA antibody levels in young outbred mice consistent with the FDA guidelines for vaccines against epidemic and pandemic influenza.
流感疫苗的生产在分离出流行毒株并使用传染性病毒后至少需要6个月时间。流行的流感病毒的血凝素（保护性抗原）变异迅速，需要重新配制疫苗。我们的目标是消除使用传染性病毒的风险，并显著缩短生产时间。以病毒RNA为模板，通过逆转录聚合酶链反应（RT-PCR）制备了编码甲型流感病毒A/越南/1203/2004（H5N1）血凝素（HA）基因的cDNA片段。重组HA（rHA）蛋白在大肠杆菌中产生，并通过尿素溶解和镍离子柱色谱从分离的包涵体中纯化。通过用福尔马林处理rHA、吸附到明矾上或两者兼用，制备候选疫苗。将候选疫苗以两周的间隔皮下注射到小鼠体内两到三次。在最后一次注射后1周收集血清，并通过酶联免疫吸附测定（ELISA）和血凝抑制（HI）测定抗体。三次注射15微克明矾吸附的rHA引发了最高的抗体反应（几何平均浓度为449EU）。用福尔马林和明矾配制的5微克rHA以及5微克明矾吸附的rHA剂量分别引发了几何平均浓度为212和177EU的抗HA IgG。所有制剂的三剂在≥80%的小鼠中获得了≥40的HI滴度。我们开发了一种在适合年度和大流行性流感疫苗接种的时间范围内生产rHA的方法。使用这种方法，rHA疫苗可在3至4周内生产出来，当与明矾配制时，在年轻的远交系小鼠中诱导的HA抗体水平符合美国食品药品监督管理局（FDA）针对流行性和大流行性流感疫苗的指导原则。
